Compounds for reducing lactation and improving health

Abstract

Preferred compounds include di-n-propyl disulfide, dimethyl disulfide, diethyl disulfide, diisopropyl disulfide, di-n-butyl-disulfide, dibenzyl disulfide, diethyl sulfide, di-n-propyl sulfide, diisopropyl sulfide, di-n-butyl sulfide, diphenyl sulfide, dibenzyl sulfide, di-n-propyl trisulfide, di-n-propyl sulfone, dibenzyl thiosulfinate, dibenzyl thiosulfonate, diisopropyl thiosulfonate, di-n-propyl thiosulfonate (PTSO) and di-n-propyl thiosulfinate (PTS).

Description

[Technical field]

[0001] The present disclosure relates to compounds for reducing lactation, for improving health, for reducing the risk and / or occurrence of intramammary infections, dry-off related stress, dry-off related inflammation and / or dry-off related infections. Preferred compounds include di-n-propyl disulfide, dimethyl disulfide, diethyl disulfide, diisopropyl disulfide, di-n-butyl-disulfide, dibenzyl disulfide, diethyl sulfide, di-n-propyl sulfide, diisopropyl sulfide, di-n-butyl sulfide, diphenyl sulfide, dibenzyl sulfide, di-n-propyl trisulfide, di-n-propyl sulfone, dibenzyl thiosulfinate, dibenzyl thiosulfonate, diisopropyl thiosulfonate, di-n-propyl thiosulfonate (PTSO) and di-n-propyl thiosulfinate (PTS). [Background technology]

[0002] In the modern dairy industry, lactating animals go through controlled cycles of milking and pregnancy, with a large overlap between the two states. A "dry period" is generally induced 40-70 days before expected calving. The dry period is the period that bridges the end of one lactation cycle to the beginning of a new lactation cycle after calving. The purpose of the dry period is to stop milk production. Cows at this stage of their lactation cycle are called "dry-off cows." The dry period allows for the recovery of the mammary gland, treatment of intramammary infections, and allows for a new lactation cycle to result in high quality milk from healthy cows. It also gives the animals an opportunity to secrete colostrum for their calves after calving.

[0003] During the dry period, changes occur in the mammary gland that are important for rejuvenating new udder tissue for lactation. It also gives the cow an opportunity to eliminate pathogens that cause mastitis in the udder (Boutinaud.M, Isaka N.,Gandemer E.,Lamberton P.,Wiart S.,De Prado AI,Sordillo LM,Lollivier V.2020“Inhibiting prolactin by cabergoline accelerates mammary gland remodelling during the early dry period in dairy cows”.Journal of Dairy Science.100 (12):9789~9798).

[0004] During the 12-24 hour non-lactating period, the levels of milk proteins and the expression of cell survival genes decrease, which results in the disappearance of epithelial cells. Changes in intracellular processes and gene regulation result in a decrease in milk production until all milk production from the mammary epithelial cells ceases (Hurley, WL (1989). Mammary Gland Function During Involution and the Declining Phase of Lactation. Journal of Dairy Science. 72 (6): 1637-1646). Furthermore, the levels of milk-specific components, such as lactose and fat, also decrease, which results in an overall steep decrease in milk production. Thereafter, the mammary gland remains in a non-lactating state. After the calf is born, parenchymal tissue and other (ruminant) udder tissues re-develop within the mammary gland prior to lactation. Colostrum is produced at the end of this stage.

[0005] "Dry-off day" refers to the day when a dairy farmer stops milking and the dairy animal enters the dry period. During the dry period, the mammary gland continues to synthesize and secrete milk, which results in increased intramammary pressure that can cause pain and discomfort to the animal (e.g., cow). Milk accumulates in the alveoli and ducts of the mammary gland, causing the udder (in ruminants) to expand by 16 hours after drying off. Approximately 16 to 18 hours after drying off, intramammary pressure increases rapidly, resulting in milk leakage and a mild inflammatory response. Evidence of inflammation includes a transient increase in blood flow, an increase in the number of neutrophils in the milk, and changes in tight junctions. Intramammary pressure peaks 2 days after drying off and then declines, but is still present 4 or 6 days after the sudden dry off. During the sudden dry off, high udder pressure (in ruminants) causes the teats to open, which can result in milk leakage. In this situation, the teats are a possible entry point for harmful microorganisms, which can result in (subclinical) mastitis. The formation of a keratin plug in each teat canal is an important natural defense mechanism against intramammary infections. However, not all cows form such a keratin plug during the dry off period.

[0006] Generally, milking may be abruptly stopped when a cow's milk production falls below 15-20 liters per day. Abrupt cessation of milking is the most common method of drying off and is used by 75% of dairy farms in the United States. With this method, milking is abruptly stopped on a day determined by the expected date of calving and the corresponding length of the dry period. Abrupt drying off is usually recommended for cows that produce less than 15-18 kg of milk on the dry day. Since the density of milk is approximately 1.032 kg / liter, this equates to 14.5-17.5 liters.

[0007] The advantage of abrupt drying is that it is simpler and less labor intensive than other methods of drying. The disadvantage of abrupt drying is that the risk of mastitis increases due to leakage of milk and increased intramammary pressure during drying, which is most prevalent in high-producing cows. For example, the probability of mastitis increases by 77% for every 5 kg of milk yield over 12.5 kg at dry off. Although udder pressure increases in all cows after drying off, it is highest in high-producing cows and lowest in low-producing cows. This leads to swollen udders and increased chances of mastitis. Furthermore, abrupt drying of cows with daily milk yields of more than 25-30 kg may shorten the lying time for the three days after drying off. Shorter recumbency times increase acidosis and lameness due to reduced rumination rates and salivation.Blanket dry cow treatment (BDCT) (administration of antibiotics to all quarters / all cows regardless of their infection status) is usually recommended to reduce the risk of intramammary infection in cows during sudden dry-off, especially if the dry-off is heavy.

[0008] Dry management regimens may be used before drying off in cows producing more than 15 liters of milk per day. For example, gradual milking is a method to reduce milk yield before drying off by reducing the frequency of milking. This reduces lactation and promotes involution of the mammary gland. Several studies have investigated the effect of frequency of milking before drying off on udder health (in ruminants). The advantages of gradual milking over abrupt drying off are reduced milk yield at dry off, reduced risk of milk leakage, and reduced intramammary pressure resulting in reduced risk of new intramammary infections both during the dry off period and after calving. Although many studies support gradual milking to reduce milk yield before drying off, it has been reported that this method has the disadvantage of delaying the formation of the keratin plug at the teat end. Therefore, cows using gradual milking should be milked at least once a day to reduce the risk of mastitis. Another drawback of staged milking is that the reduction in udder size occurs slowly and there is an inflammatory reaction that negatively affects the cow's comfort. However, cows who are milked less frequently spend less time lying down (which is a sign of discomfort). However, there is some evidence that this method can still cause discomfort due to udder expansion.

[0009] Another approach to reduce milk production is gradual feeding. In 2016, the USDA reported that 82% and 18% of cows in the United States were abruptly and gradually dried off, respectively. In this method, milk production is reduced by slowing the rate of glucose transport to the mammary gland through feeding. Gradual feeding can be done in several ways, such as by removing concentrates (for 14 days before drying off), reducing dry matter intake (for 14 days), feeding low-energy diets (for 7 days before drying off), or removing hay (for 5 days before drying off). Feeding only straw can have adverse effects on cow health, such as reduced heart rate, elevated plasma cortisol levels, and elevated somatic cell counts during the dry off period. Although a graded feeding regime can be effective in reducing milk yield before drying off, extreme metabolic stress due to the degree of feed restriction during the feeding period of the graded feeding regime should be avoided.

[0010] The advantage of staged feeding is the reduction in milk production at dry off, which is associated with a reduced risk of new intramammary infections. Staged feeding may be preferred over staged milking in some cases because of the reduced milk yield. Staged feeding induces a reduction in milk yield through reduced nutrition to the mammary gland, rather than through the mechanism of udder pressure, which promotes udder filling. It has been reported that cows with reduced dry matter intake have less than half the milk leakage compared to cows that are suddenly dried off.

[0011] Therefore, it is estimated that the risk of mastitis due to milk leakage is lower with staged feeding compared to staged milking. However, this method also has disadvantages. Staged feeding due to reduced dry matter intake may increase the stress level of the cow. This stress may result in a weakened immune system and a poor energy balance. In addition, there is concern about the negative impact on the birth weight of the calf if the nutritional deficiency is too great and if it lasts longer than 80-90 days during the gestation period. Another disadvantage of staged feeding is the additional labor required. As a result, staged feeding is less feasible for small dairy farms, where feeding cows a lot of mixed ration is often problematic.

[0012] Despite considerable research efforts, it has been difficult to develop a universal management method to reduce milk production without side effects on animal health or welfare (Martin et al., 2020. Automated gradual reduction of milk production before dry-off: Effects on udder health, involution and inner teat morphology. Livestock Science, 233, 103942).

[0013] The reduction of lactation in mammals is a painful process. This is especially true for mammals that undergo this process frequently. Drying off of cows is a risky, painful and stressful period. The mammary glands continue to secrete milk during early involution, resulting in elevated udder pressure (in ruminants) and milk leakage. This increases the risk of intramammary infections, which can subsequently result in (subclinical) mastitis, discomfort, pain and stress in the cow. The higher the milk production of the cow, the higher the risk of mastitis and the greater the pain and stress.

[0014] The health of the udder plays an essential role for dairy animals, both from a health and wellness point of view as well as from an economic point of view. Infection in the mammary gland of dairy animals, e.g. cows, known as mastitis, has a major economic impact on dairy farmers worldwide. Several factors are known to upset the balance at the level of the udder, which may impair the ability of the dairy animal to kill the microorganisms that cause mastitis. As a result, the host's response mechanisms are unable to mount an efficient defense response to eliminate the invading pathogen, leading to bacterial colonization of the udder and the development of clinical or subclinical mastitis. Bacterial colonization, especially the formation of bacterial reservoirs, in the udder of dairy cows generally leads to infections that are difficult to combat and are treated with antibiotics.

[0015] Drying off is the abrupt cessation of milking which initiates involution (the state of the mammary gland changing from lactating to non-lactating). After the dry off date, when milk accumulates in the udder, the udder becomes susceptible to new udder infections. The reason for this susceptibility is thought to be the shortening of the teat canal due to udder pressure and the incomplete formation of the keratin plug.

[0016] During the dry period of dairy cows, new intramammary infections occur in approximately 10% to 17% of the udders (ruminant animals), which may lead to subclinical mastitis (Pantoja at al. 2009. Somatic cell count status across the dry period as a risk factor for the development of clinical mastitis in the subsequent lactation. Journal of dairy science, 92(1), 139-148). Infection during this period increases the likelihood of developing mastitis in the following lactation (Capuco et al, 1997. A study of the incidence and significance of intramammary enterobacterial infections acquired during the dry period. Journal of dairy science, 83(9), 1957-1965). Mastitis reduces milk production, deteriorates milk quality, and leads to relatively large economic losses (Hertl et al., 2014. Pathogen-specific effects on milk yield in repeated clinical mastitis episodes in Holstein dairy cows. Journal of dairy science, 97(3), 1465-1480) and veterinary costs. Therefore, it is important to prevent intramammary infection.

[0017] Treatment with topical antibiotics (AB) at the beginning of the dry period reduces (subclinical) mastitis but rarely solves the problem due to the formation of a biofilm in which the bacteria are in a dormant stage and relatively less susceptible to antibiotics. Scherpenzeel et al. found that the incidence of clinical mastitis was 1.7 times higher in udders dried without antibiotics compared to udders dried with antibiotics in dry cows without antibiotics compared to udders dried with antibiotics (Scherpenzeel et al., 2014. Evaluation of the use of dry cow antibiotics in low somatic cell count cows. Journal of Dairy Science, 97(6), 3606~3614). Since the use of antibiotics is permitted in many countries only for therapeutic purposes to prevent antibiotic resistance by microorganisms, it is important to find other methods of quality dry period management to reduce the incidence of mastitis in subsequent lactations.

[0018] Dry cows represent an important perspective regarding the future profitability of a dairy farm. Proper care, feeding and management of dry cows helps to improve milk production and udder health during the next lactation. In contrast, poor dry cow management practices can result in a 1,100 liter reduction in milk production. Proper management of the dry period is therefore important to have a productive and profitable dairy herd.

[0019] In humans, the lactation cycle begins at conception. Delivery of the placenta triggers the transition to lactation, followed by continued low levels of synthesis that require periodic milk removal. The lactation cycle is completed after the infant is weaned. Summary of the Invention [Problem to be solved by the invention]

[0020] One object of the present disclosure is to provide compounds and treatments useful in reducing lactation in mammals, as well as compounds and treatments for use in drying off lactating dairy animals. [Means for solving the problem]

[0021] The present disclosure provides compounds having formula I, particularly di-n-propyl disulfide, for use in therapy. In particular, such compounds are useful for reducing lactation, preventing intramammary infections, and reducing the risk of stress, inflammation, or infection during weaning / drying. The following are preferred embodiments of the present disclosure.

[0022] 1. A compound of formula I below or a composition comprising said compound for use in reducing lactation in a mammal: [ka] Formula I Here, R 1 and R 2 is independent, C 1~4 alkyl and phenyl; 1~4 Alkyl is methyl, ethyl, n-propyl or n-butyl; 1~4 The alkyl and phenyl are unsubstituted.

[0023] 2. The compound or composition for use according to item 1, wherein the mammal is a ruminant, preferably a cow.

[0024] 3. A compound according to formula I below or a composition comprising said compound for use in the prophylactic treatment of intramammary infections and / or for use in reducing the incidence of dry-off associated stress, dry-off associated inflammation or dry-off associated infection: [ka] Formula I Here, R 1and R 2 is independent, C 1~4 alkyl and phenyl; 1~4 Alkyl is methyl, ethyl, n-propyl or n-butyl; 1~4 The alkyl and phenyl are unsubstituted.

[0025] 4. A compound or composition of paragraph 3, wherein milking is stopped suddenly or gradually in said mammal and said compound is administered before or on the day said milking is stopped.

[0026] 5. A compound according to formula I below or a composition comprising said compound for use in promoting the health and well-being of a lactating mammal: [ka] Formula I Here, R 1 and R 2 is independent, C 1~4 alkyl and phenyl; 1~4 Alkyl is methyl, ethyl, n-propyl or n-butyl; 1~4 The alkyl and phenyl are unsubstituted.

[0027] 6.R 1 and R 2 The compound or composition for use according to any one of items 1 to 5, wherein are identical.

[0028] 7. The compound or composition for use according to any one of items 1 to 6, wherein the compound is selected from di-n-propyl disulfide, dimethyl disulfide, diethyl disulfide, di-n-butyl-disulfide and diphenyl disulfide.

[0029] 8. The compound or composition for use according to any one of items 1 to 7, wherein said use is in combination with dry cow therapy.

[0030] 9. The compound or composition for use according to any one of clauses 1 to 8, further comprising administering a prolactin inhibitor, such as cabergolin and quinagolide; casein hydrolysate; or an acidogenic mineral bolus.

[0031] 10. The compound or composition for use according to any one of clauses 1 to 9, further comprising the administration of an antibiotic, antifungal or anti-inflammatory agent.

[0032] 11. A method comprising administering to a mammal or lactating mammal a pharmaceutical or veterinary composition or a nutraceutical composition comprising a compound according to formula I: [ka] Formula I Here, R 1 and R 2 is independent, C 1~4 alkyl and phenyl; 1~4 Alkyl is methyl, ethyl, n-propyl or n-butyl; 1~4 The alkyl and phenyl are unsubstituted. Preferably, the method is for reducing lactation in a mammal, and administration of the compound or composition results in reduced lactation. Preferably, the method is for prophylactic treatment of intramammary infections and / or for reducing the incidence of dry-off-related stress, dry-off-related inflammation or dry-off-related infection in a mammal, and administration of the compound or composition results in prophylactic treatment of intramammary infections and / or for reducing the incidence of dry-off-related stress, dry-off-related inflammation or dry-off-related infection. Preferably, the method is for use in promoting the health and well-being of a lactating mammal, and administration of the compound or composition results in improving or maintaining the health and well-being of a lactating mammal. 12. The compound or composition for use according to any one of clauses 1 to 11, wherein the compound is formulated as a single dosage unit containing at least 50 grams, preferably at least 70 grams, of the compound, and the composition is administered to a pregnant ruminant.

[0033] 13. A method according to any one of claims 1 to 12, or a compound or composition for use therein, comprising selecting a pregnant cow that produces at least 10 liters of milk per day, and administering the composition to the cow.

[0034] 14. The method or compound or composition for use according to any one of items 1 to 13, wherein milking is stopped suddenly or gradually in the mammal and the compound is administered before or on the day that milking is stopped.

[0035] 15. A pharmaceutical or veterinary composition or a functional food composition comprising a compound according to formula I: [ka] Formula I Here, R 1 and R 2 is independent, C 1~4 alkyl and phenyl; 1~4 Alkyl is methyl, ethyl, n-propyl or n-butyl; 1~4 The alkyl and phenyl are unsubstituted.

[0036] 16. The composition according to paragraph 15, wherein the compound is formulated as a single dosage unit comprising at least 50 grams of the compound, preferably at least 70 grams of the compound.

[0037] 17.R 1 and R 2 Item 17. The composition according to item 15 or 16, wherein:

[0038] 18. The composition according to item 17, wherein the compound is selected from di-n-propyl disulfide, dimethyl disulfide, diethyl disulfide, di-n-butyl disulfide and diphenyl disulfide.

[0039] 19. The composition according to any one of items 15 to 18, further comprising a dry-off agent, preferably selected from a prolactin inhibitor, such as cabergolin and quinagolide; casein hydrolysate; or an acidogenic mineral bolus.

[0040] 20. The composition according to any one of items 15 to 19, wherein the composition further comprises PTSO.

[0041] 21. The composition according to any one of items 15 to 20, wherein the composition is a gel capsule.

[0042] The following are also preferred embodiments of the present disclosure.

[0043] 1.a) In reducing lactation in mammals; b) in the prophylactic treatment of intramammary infections and / or in reducing the incidence of dry-off-related stress, dry-off-related inflammation or dry-off-related infection in mammals; and / or c) in promoting the health and well-being of lactating mammals A compound according to formula II below or a composition comprising said compound for use: [ka] Formula II Where: R 1 and R 2 is independent, C 1~4 Selected from the group consisting of alkyl, phenyl and benzyl; Q 1is selected from the group consisting of -SS-, -S-, -SSS-, -S(O)2-, -S(O)-S- and -S(O)2-S-; With the proviso that the compound according to formula II is not diphenyl disulfide; Here, preferably, R 1 and R 2 is independent, C 1~4 selected from the group consisting of alkyl and benzyl; Preferably, the C 1~4 Alkyl is methyl, ethyl, n-propyl, isopropyl or n-butyl; Preferably, the C 1~4 The alkyl and benzyl are unsubstituted.

[0044] 2.a) In reducing lactation in mammals; b) in the prophylactic treatment of intramammary infections and / or in reducing the incidence of dry-off-related stress, dry-off-related inflammation or dry-off-related infection in mammals; and / or c) in promoting the health and well-being of lactating mammals A compound according to formula I below or a composition comprising said compound for use: [ka] Formula I Here, R 1 and R 2 is independent, C 1~4 is selected from the group consisting of alkyl and benzyl; 1~4 Alkyl is methyl, ethyl, n-propyl, isopropyl or n-butyl; 1~4 The alkyl and benzyl are unsubstituted.

[0045] 3. The compound or composition for use according to embodiment 1 or 2, wherein the mammal is a ruminant, preferably a cow.

[0046] 4.R 1 and R2 and are identical. The compound or composition for use according to any one of embodiments 1 to 3.

[0047] 5. The compound or composition for use according to any one of the preceding embodiments, wherein the compound is selected from di-n-propyl disulfide, dimethyl disulfide, diethyl disulfide, diisopropyl disulfide, di-n-butyl-disulfide and dibenzyl disulfide.

[0048] 6. The compound or composition for use according to any one of embodiments 1 to 5, wherein the compound is di-n-propyl disulfide.

[0049] 7. The compound or composition for use according to any one of embodiments 1 or 3-4, wherein the compound is selected from di-n-propyl sulfide, diethyl sulfide, diisopropyl sulfide, di-n-butyl sulfide, diphenyl sulfide, dibenzyl sulfide, di-n-propyl trisulfide, di-n-propyl sulfone, dibenzyl thiosulfinate, dibenzyl thiosulfonate, diisopropyl thiosulfonate, di-n-propyl thiosulfonate (PTSO) and di-n-propyl thiosulfinate (PTS).

[0050] 8. The compound or composition for use according to any one of the preceding embodiments, wherein said use is in combination with dry cow therapy.

[0051] 9. The compound or composition for use according to any one of embodiments 1 to 8, further comprising administering a prolactin inhibitor, such as cabergoline and quinagolide; a casein hydrolysate; or an acidogenic mineral bolus.

[0052] 10. The compound or composition for use according to any one of the preceding embodiments, wherein said use further comprises the administration of an antibiotic, an antifungal or an anti-inflammatory agent.

[0053] 11. A method for reducing lactation in a mammal; for the prophylactic treatment of intramammary infections and / or for reducing the occurrence of dry-off related stress, dry-off related inflammation or dry-off related infections in a mammal; or for promoting the health and well-being of a lactating mammal, said method comprising administering to the lactating mammal a pharmaceutical or veterinary composition or a functional food composition comprising a compound according to formula II below: [ka] Formula II Where: R 1 and R 2 is independent, C 1~4 Selected from the group consisting of alkyl, phenyl and benzyl; Q 1 is selected from the group consisting of -SS-, -S-, -SSS-, -S(O)2-, -S(O)-S- and -S(O)2-S-; With the proviso that the compound according to formula II is not diphenyl disulfide; Here, preferably, R 1 and R 2 is independent, C 1~4 selected from the group consisting of alkyl and benzyl; Preferably, the C 1~4 Alkyl is methyl, ethyl, n-propyl, isopropyl or n-butyl; Preferably, the C 1~4 The alkyl and benzyl are unsubstituted.

[0054] 12. The composition according to embodiment 11, wherein the compound is according to formula I: [ka] Formula I Here, R 1and R 2 is independent, C 1~4 alkyl and benzyl; 1~4 Alkyl is methyl, ethyl, n-propyl, isopropyl or n-butyl; 1~4 The alkyl and benzyl are unsubstituted.

[0055] 13. The compound or composition for use according to any one of embodiments 1-12, wherein the compound is formulated as a single dosage unit comprising at least 50 grams of the compound, preferably at least 70 grams of the compound, and wherein the composition is administered to a pregnant ruminant.

[0056] 14. The method or compound or composition for use according to any one of embodiments 1 to 13, comprising selecting a pregnant cow producing at least 10 liters of milk per day and administering the composition to said cow.

[0057] 15. A compound or composition for use according to any one of embodiments 1-14, wherein milking is stopped suddenly or gradually in the mammal and the compound is administered before or on the day that milking is stopped.

[0058] 16. A pharmaceutical or veterinary composition or a functional food composition comprising a compound according to formula II: [ka] Formula II Where: R 1 and R 2 is independent, C 1~4 Selected from the group consisting of alkyl, phenyl and benzyl; Q 1 is selected from the group consisting of -SS-, -S-, -SSS-, -S(O)2-, -S(O)-S- and -S(O)2-S-; With the proviso that the compound according to formula II is not diphenyl disulfide; Here, preferably, R 1 and R 2 is independent, C 1~4 selected from the group consisting of alkyl and benzyl; Preferably, the C 1~4 Alkyl is methyl, ethyl, n-propyl, isopropyl or n-butyl; Preferably, the C 1~4 The alkyl and benzyl are unsubstituted.

[0059] 17. The compound is according to formula I: [ka] Formula I Here, R 1 and R 2 is independent, C 1~4 alkyl and benzyl; 1~4 Alkyl is methyl, ethyl, n-propyl, isopropyl or n-butyl; 1~4 The alkyl and benzyl are unsubstituted. 17. The composition of embodiment 16.

[0060] 18. The composition according to embodiment 16 or 17, wherein the compound is formulated as a single dosage unit comprising at least 50 grams of the compound, preferably at least 70 grams of the compound.

[0061] 19.R 1 and R 2 The composition of any one of embodiments 16 to 18, wherein

[0062] 20. The composition according to embodiment 19, wherein the compound is selected from di-n-propyl disulfide, dimethyl disulfide, diethyl disulfide, diisopropyl disulfide, di-n-butyl-disulfide, dibenzyl disulfide, diethyl sulfide, di-n-propyl sulfide, diisopropyl sulfide, di-n-butyl sulfide, diphenyl sulfide, dibenzyl sulfide, di-n-propyl trisulfide, di-n-propyl sulfone, dibenzyl thiosulfinate, dibenzyl thiosulfonate, diisopropyl thiosulfonate, di-n-propyl thiosulfonate (PTSO) and di-n-propyl thiosulfinate (PTS).

[0063] 21. The composition according to embodiment 19, wherein the compound is di-n-propyl disulfide.

[0064] 22. The composition of any one of embodiments 16 to 21, further comprising a dry-off agent, preferably selected from a prolactin inhibitor, such as cabergoline and quinagolide; a casein hydrolysate; or an acid-generating mineral bolus.

[0065] 23. The composition of any one of embodiments 16 to 22, wherein the composition is a gel capsule.

[0066] Such compositions are useful in any of the methods or uses disclosed herein. [Brief description of the drawings]

[0067] [Figure 1] Figure 1 shows the average milk production of 8 placebo cows from Example 1. Cows were treated with a placebo capsule on day t=13. [Diagram 2] Figure 2 shows milk leakage. The upper lines represent milk leakage as reported in the previous literature (dark grey line) and the control of this experiment (grey line) in which cows produced more than 12.5 kg of milk per day. The bar graph shows the % of cows with milk leakage at certain times after sudden drying off. [Diagram 3] Figure 3 shows udder pressure (ruminants). The upper dark grey line and the lower grey line respectively represent udder pressures reported in previous literature and control observations in this study using cows that produced more than 12.5 kg of milk per day. The lower dark grey line shows the average udder pressure at each fixed time after sudden drying off for cows treated with synthetic DPD. [Figure 4] Figure 4 shows stress. The upper line shows the predicted stress of cows as given in the literature (gray line) and the control of this experiment (dark grey) using cows that produced more than 12.5 kg of milk per day at dry off. The lower line shows the average stress of cows treated with DPD at each fixed time after sudden dry off. [Diagram 5] Figure 5 shows milk leakage. The bar graph shows the percentage of cows with milk leakage at each fixed time after sudden drying off for DPD enriched OE treated cows. The line shows the expected milk leakage. [Figure 6] Figure 6 shows udder pressure (ruminants). The lower line shows the average udder pressure (ruminants) at each fixed time after a quick dry off. The upper line shows the predicted udder pressure (ruminants) for cows with over 12.5 kg per day at dry off. [Figure 7] Figure 7 shows stress. The lower line shows the average stress behavior level at each fixed time after sudden dry off for cows treated with DPD enriched OE. The upper line shows the predicted stress for cows with more than 12.5 kg per day at dry off. [Figure 8]Figure 8 shows udder pressure (ruminants). The left bar graph shows udder pressure (ruminants) before the last milking and the lower line shows udder pressure (ruminants) (g / m2) of the three test cows at fixed times. The upper line is the data for the control group. The right bar graph shows udder pressure (ruminants) before the last milking and the upper line shows udder pressure (ruminants) (g / m2) of the three test cows at fixed times. [Figure 9-1] Figure 9 shows DPD treatment and incidence of mastitis. Legend: vid - DPD enriched OE treated group; dol-DC Liquid treated group; AB - antibiotic treatment. [Figure 9-2] Figure 9 shows DPD treatment and incidence of mastitis. Legend: vid - group treated with DPD enriched OE; dol - group treated with DC liquid; AB - antibiotic treatment. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0068] The present disclosure provides compounds useful for a number of methods, and those of skill in the art will appreciate that references to the use, administration, etc. of a compound also include the use, administration, etc. of a composition comprising the compound.

[0069] The present disclosure provides compounds disclosed herein and compositions comprising the compounds useful for reducing lactation in mammals. As used herein, "lactation" refers to the secretion of milk by the mammary gland. Thus, the compounds disclosed herein are useful for reducing milk production. In some embodiments, milk production is reduced by at least 10% compared to milk production before treatment. In some embodiments, the comparison is performed between the first day before treatment and the first day after treatment.

[0070] The compounds disclosed herein and compositions comprising the compounds are useful in the prophylactic treatment of intramammary infections, such as mastitis.

[0071] One of the important factors for the subclinical occurrence of mastitis is high milk production before the dry off period. One study demonstrated that cows with milk yields of more than 5 kg just before the start of the dry off period are three times more likely to develop new (subclinical) mastitis compared to cows with low milk yields (Dingwell et al., 2001. Impact of milk production and important management factors on the process of dry-off in lactating dairy cows, Dairy Day 2001, p. 27). Another study showed that for every 5 kg increase in milk yield above 12.5 kg, the probability of having environmental (subclinical) mastitis at calving increases by 77% (Rajala-Schultz et al., 2005. Association between milk yield at dry-off and probability of intramammary infections at calving. Journal of Dairy Science, 88(2), 577-579). High milk production can lead to high udder pressure and increased milk leakage (Summers et al., 2004. Influence of feeding level after drying off on incidence of mastitis and keratin plug formation in dairy cows. Proceedings of the New Zealand Society of Animal Production 2004, Vol 64). Milk leakage during the dry period increases the chances of developing clinical mastitis during the dry period by four times (Schukken et al., 1993. A randomized blind trial on dry cow antibiotic infusion in a low somatic cell count herd. Journal of Dairy Science, 76(10), 2925-2930).Studies have demonstrated that cows with high milk yields before the start of the dry period have a higher percentage of teat opening during the second and third dry weeks and a higher probability of developing (subclinical) mastitis immediately after calving compared to cows with low milk yields at the start of the dry period (Odensten et al., 2007. Metabolism and udder health at dry-off in cows of different breeds and production levels. Journal of Dairy Science, 90(3), 1417-1428). Closed or sealed teat canals are less likely to develop mastitis during the dry period than open teat canals (Williamson et al., 1995. The prophylactic effect of a dry-cow antibiotic against Streptococcus uberis. New Zealand veterinary journal, 43(6), 228-234). Therefore, rapid development of the keratin plug after the onset of the dry period is important to prevent new intramammary infections during the dry period (Lacy-Hulbert et al., 1999. Sealing of the bovine teat canal after drying off. In Proceeding-New Zealand Society of Animal Production (Vol.59, pp.198-200). This keratin plug forms a physical barrier and thus prevents bacteria from entering the teat canal. A low milk yield at the onset of the dry period is beneficial to minimize milk leakage and promote the formation of the keratin plug. This prevents intramammary infections during the dry period and the resulting new (subclinical) mastitis.

[0072] As used herein, "prophylactic treatment of intramammary infection" refers to reducing the likelihood of intramammary infection in the breast or udder (of ruminants) and / or reducing the severity and / or duration of symptoms due to infection. Preferably, the treatment is for a mammal. Preferably, the treatment results in the maintenance of health of the individual.

[0073] The compounds and compositions comprising the compounds disclosed herein are useful in reducing the incidence of infections, particularly dry-off related infections, or infections associated with weaning.

[0074] Infections can be caused by a variety of pathogens, most notably bacteria and viruses. In udder infections (in ruminants), other microorganisms are also involved, such as micro-algae (e.g. Prototeca spp., Mycoplasma spp.), viruses, yeasts and fungi. The mammalian host responds to infection with an innate response, often accompanied by inflammation, followed by an adaptive response.

[0075] The most prominent microorganisms which can invade the udder (of ruminants) and cause bovine mastitis are the bacteria Staphylococcus aureus, Streptococcus uberis, Streptococcus agalactia, Streptococcus dysgalactiae, as well as Serratia marcescens, Leptospira spp., Pseudomonas spp., Brucella spp., Escherichia coli, Klebsiella spp., Mycobacterium spp., and other strains of bacteria. spp. and other facultative pathogenic Enterobacteriaceae; and the micro-algae Prototheca spp. Examples of viruses that may invade the mammary gland and directly or indirectly cause mastitis are bovine herpesvirus 1, bovine herpesvirus 2, vaccinia, bovine viral diarrhoea, virus cowpox, pseudocowpox, vesicular stomatitis, foot-and-mouth disease viruses, bovine papillomaviruses, bovine immunodeficiency virus, parainfluenza 3, bovine leukaemia virus infections may play an (indirect) role in the pathogenesis of bovine mastitis.These viruses induce, for example, teat lesions in the teat canal, which results in a weakening of the natural defense mechanisms of the udder (in ruminants) and indirectly in bovine mastitis due to bacterial pathogens. Moreover, during the dry period, the possibility of potentially pathogenic yeasts, protozoa and fungi, such as Candida spp., Cryptococcus spp., Rhodotorula spp., Stephanoascus spp., Trichosporum spp. and Kodamaea spp., Kloeckeria spp. and Aspergillus spp. and Neospora spp., also increases for their invasion of the teat.

[0076] The compounds disclosed herein and compositions containing the compounds are useful in reducing the occurrence of inflammation, particularly dry-off-related inflammation or inflammation associated with weaning. Inflammation is part of the complex biological response of body tissues to (harmful) stimuli, such as pathogens and reduced or low milk production, and is a defense response involving immune cells and molecular mediators. The function of inflammation is to eliminate pathogens.

[0077] During the dry period or during weaning, infections (e.g., udder infections in cows) can occur, resulting in inflammation of tissues. Activated immune cells and inflammatory responses can also damage tissues, such as those in the milk gland. Therefore, inhibiting the inflammatory response can prevent or reduce damage to tissues. For example, there will be less damage to the milk gland and the cow will recover milk production more quickly.

[0078] The compounds disclosed herein and compositions comprising the compounds are useful in reducing the incidence of stress, particularly dry-off related stress or stress associated with weaning.

[0079] Although such use is prophylactic, one of skill in the art will recognize that prophylaxis is not usually a 100% reduction, rather, there is a reduction in likelihood compared to individuals not treated with the compounds of the invention.

[0080] The compounds disclosed herein and compositions containing the compounds are useful for promoting the health and / or well-being of lactating mammals, especially when a reduction in milk production is desired. As mentioned above, the weaning and drying process can be painful, stressful, and carries the risk of intramammary infection. The compounds disclosed herein have a positive effect during the weaning and drying process. In addition, without wishing to be bound by theory, the compounds disclosed herein may have an effect on serotonin, oxytocin, prolactin, and / or dopamine.

[0081] The compound or composition may be administered to the mammal in conjunction with cessation of milk production, as further described herein, In particular, the compound or composition may be administered before or on the day that milk production is stopped (suddenly or gradually) in the mammal.

[0082] The compounds and compositions comprising the compounds disclosed herein can be administered to any mammal, preferably a lactating mammal. In some embodiments, the mammal is a human. In some embodiments, the mammal is a non-human mammal. Preferably, the mammal is a ruminant (e.g., cows and goats), more preferably a cow. In some embodiments, the mammal is pregnant. In certain embodiments, the compounds are administered to a pregnant lactating mammal to reduce lactation before parturition. In some embodiments, the mammal is a cow, and preferably cows with milk production of more than 10 L / day, preferably more than 11.5 L / day, are selected for treatment.

[0083] The present disclosure also provides an in vitro use of a compound disclosed herein to study the effect on mammalian, preferably ruminant, bovine or human, mammary tissue. Preferably, the method is for reducing milk formation in cultured mammary gland tissue on a surface. In some embodiments, the method comprises contacting the tissue attached to a surface with a composition disclosed herein.

[0084] The present disclosure relates to compounds according to Formula I below: [ka] Formula I Here, R 1 and R 2 is independent, C 1~4 In some embodiments, R is selected from the group consisting of alkyl and phenyl. 1 and R 2 is independent, C 1~4 In a preferred embodiment, R 1 and R 2 are identical.

[0085] The present disclosure further relates to compounds according to Formula II: [ka] Formula II Where: R 1 and R 2 is independent, C 1~4 Selected from the group consisting of alkyl, phenyl and benzyl; Q 1 is selected from the group consisting of -SS-, -S-, -SSS-, -S(O)2-, -S(O)-S- and -S(O)2-S-; With the proviso that the compound according to formula II is not diphenyl disulfide. In some embodiments, the compound according to formula II is not diethyl sulfide. In preferred embodiments, R1 and R 2 is independent, C 1~4 In a preferred embodiment, R 1 and R 2 are identical.

[0086] As used herein, "alkyl" refers to a saturated aliphatic hydrocarbyl group. Unless otherwise specified, the alkyl group can be linear or branched. Preferably, the alkyl group is linear. As used herein, the alkyl group can be substituted or unsubstituted. Preferably, the alkyl group is unsubstituted. Preferably, the C 1~4 Alkyl is methyl, ethyl, n-propyl, isopropyl or n-butyl.

[0087] As used herein, "substituted" indicates that a group contains one or more substituents. Preferably, the substituents are independently selected from halogen atoms, -C(O)OH, -C(O)NH, -OH, =O, C 1~3 Preferably, the halogen atom is selected from the group consisting of -Cl, -F, -Br and -I. Most preferably, the halogen is -Cl. In preferred embodiments, the groups as disclosed herein contain no more than three substituents, more preferably no more than two substituents, and most preferably no more than one substituent. Preferably, C 1~4 Alkyl and phenyl are unsubstituted. 1~4 The alkyl and benzyl are unsubstituted.

[0088] In a preferred embodiment, the compound according to formula I is selected from di-n-propyl disulfide, dimethyl disulfide, diethyl disulfide, di-n-butyl-disulfide and diphenyl disulfide. In a preferred embodiment, the compound according to formula I or formula II is selected from the group consisting of di-n-propyl disulfide, dimethyl disulfide, diethyl disulfide, diisopropyl disulfide, di-n-butyl-disulfide and dibenzyl disulfide. In a preferred embodiment, the compound of the present invention is selected from the compounds listed in Table 18.

[0089] In a preferred embodiment, the compound according to Formula I or Formula II is di-n-propyl disulfide.

[0090] In a preferred embodiment, the compound according to formula II is selected from the group consisting of diethyl sulfide, di-n-propyl sulfide, diisopropyl sulfide, di-n-butyl sulfide, diphenyl sulfide, dibenzyl sulfide, di-n-propyl trisulfide, di-n-propyl sulfone, dibenzyl thiosulfinate, dibenzyl thiosulfonate, diisopropyl thiosulfonate, di-n-propyl thiosulfonate (PTSO) and di-n-propyl thiosulfinate (PTS).

[0091] In some embodiments, the compound is obtained from natural sources, such as plants. Compounds can be extracted from plant materials in various ways. The appropriate method depends on the chemical nature of the compound. For example, extraction can start with a non-polar solvent, and then can be extracted with a solvent of increasing polarity. Compounds can also be prepared synthetically.

[0092] In a preferred embodiment, the compound according to Formula I or Formula II is selected from the group consisting of di-n-propyl disulfide (DPD; CAS#629-19-6), dimethyl disulfide (DMDS CAS#624-92-0), diethyl disulfide (CAS#110-81-6), diisopropyl disulfide (CAS#4253-89-8), di-n-butyl-disulfide (CAS#629-45-8), diphenyl disulfide (CAS#882-33-7) and dibenzyl disulfide (CAS#150-60-7). In a preferred embodiment, the compound according to formula II is diethyl sulfide (CAS#352-93-2), di-n-propyl sulfide (CAS#111-47-7), diisopropyl sulfide (CAS#625-80-9), di-n-butyl sulfide (CAS#544-40-1), diphenyl sulfide (CAS#139-66-2), dibenzyl sulfide (CAS#538-74-9), di-n-propyl trisulfide (CAS#6028-61-1), ), di-n-propyl sulfone (CAS#598-03-8), dibenzyl thiosulfinate (CAS#16302-98-0), dibenzyl thiosulfonate (CAS#16601-40-4), diisopropyl thiosulfonate (CAS#10027-69-7), di-n-propyl thiosulfonate (PTSO; CAS#1113-13-9) and di-n-propyl thiosulfinate (PTS; CAS#1948-52-3). These compounds are commercially available, for example, from Sigma-Aldrich.

[0093] In a preferred embodiment, the compound is di-n-propyl disulfide (DPD). As described in the examples, DPD has surprisingly been shown to reduce milk production without any obvious toxic side effects. A dose of 200 grams of di-n-propyl disulfide was administered to cows, and no side effects were observed. In the examples described herein, 80 ml of pure di-n-propyl disulfide reduced milk production similarly to a low dose of 20 ml of DPD (Example 2). The density of di-n-propyl disulfide is 0.96 g / ml (20° C.). Thus, 80 ml of pure di-n-propyl disulfide is equivalent to 77 g.

[0094] Exemplary doses of the compound according to Formula II for cattle are shown in Table 17 (Example 8) in milliliters (ml) or grams (g). In a preferred embodiment, the minimum dose of the compound according to Formula II is at least 30% of the exemplary dose. For example, the minimum dose of diethyl disulfide is at least 18.9 ml. In a preferred embodiment, the minimum dose of the compound according to Formula II is at least 50% from the exemplary dose. For example, the minimum dose of diethyl disulfide is at least 31.5 ml. In a preferred embodiment, the minimum dose of the compound according to Formula II is at least 80% from the exemplary dose. For example, the minimum dose of diethyl disulfide is at least 50.4 ml. In some embodiments, the maximum dose is 200% or 300% of the exemplary doses shown in Table 17.

[0095] The actual dose level of the compounds described herein can be varied to obtain an amount of active ingredient that is effective for achieving the desired therapeutic response for a particular individual, composition, and mode of administration and is not toxic to the individual. The selected dose level depends on various factors, including, for example, the activity of the particular compound, the route of administration, the time of administration, the rate of excretion of the particular compound used, the duration of treatment, other drugs, compounds and / or materials used in combination, the age, sex, weight, condition, general health and previous medical history of the individual being treated, and similar factors well known in the medical art. A physician or veterinarian having ordinary skill in the art can easily determine and prescribe the effective amount of the compound required.

[0096] In some embodiments, the mammal is administered at least 0.2 g, preferably at least 0.5 g, of a compound as disclosed herein per day. In an exemplary embodiment, the cow is administered at least 50 g, preferably at least 60 g, more preferably at least 70 g, of a compound as disclosed herein (preferably DPD). In some embodiments, the cow is administered at least 75 grams or at least 77 grams of a compound as disclosed herein (preferably DPD). In some embodiments, the cow is administered 50-200 grams, preferably 70-200 grams, more preferably 75-200 grams of a compound as disclosed herein (preferably DPD). Preferably, the cow is administered at least 77 grams of a compound as disclosed herein (preferably DPD). Preferably, the cow is administered approximately 77 grams of a compound as disclosed herein (preferably DPD). Such dosages may be provided as single dosage units.

[0097] It is clear to those skilled in the art that smaller amounts of compound can be administered to small animals. The examples describe administering a tablet containing 77 g of DPD to cows. The average body weight of a cow is approximately 650 kg, which corresponds to a dose of about 118 mg of DPD / kg. Those skilled in the art recognize that small animals have a higher metabolic rate and therefore require a higher drug dose on a body weight basis. Dose conversion between animals and between humans and animals is discussed in Nair and Jacob (J Basic Clin Pharm. March 2016-May 2016;7(2):27-31) and Holliday et al. (1967 The Relation of Metabolic Rate to Body Weight and Organ Size. A Review. Pediat. Res. 1:185-195).

[0098] Without wishing to be bound by theory, the present disclosure provides that the compounds disclosed herein can have beneficial effects after a single dose. In a preferred embodiment, the effect is achieved by giving a single dose of the compounds disclosed herein. As demonstrated in the examples, a single dose results in a (reversible) reduction in milk production. Combining with other strategies, such as drying off or weaning, can result in a further, more durable reduction in milk production. In an exemplary embodiment, the compounds disclosed herein are given to lactating pregnant cows, and the cows are not milked until the calf is born. Once the new calf is born, milk production resumes.

[0099] The present disclosure also provides for multiple administrations. For example, the composition may be provided one or more times a day, daily, weekly, or monthly. In exemplary embodiments, the composition may be provided once a week until milk production ceases or is significantly reduced. In some embodiments, the composition may be provided once every 3-4 days or once every 2 days.

[0100] The compound is particularly useful when provided systemically (e.g., orally).As further disclosed herein, the compound is preferably provided as a pharmaceutical or veterinary composition, or as a functional food.As will be appreciated by those skilled in the art, such compositions are suitable for administration to humans and other animals.

[0101] In some embodiments, the compositions disclosed herein are provided as functional food compositions. As used herein, the term "functional food" refers to foods that are prepared not only for their nutritional properties but also to perform a specific function, such as improving health or reducing the risk of contracting a disease. Such functional foods may also be referred to as dietary supplements or (animal) food additives. For this purpose, biologically active compounds may be added to them, such as minerals, vitamins, fatty acids, bacteria with beneficial effects, dietary fiber, and antioxidants. Such foods may be in any form suitable for oral intake, such as liquid, gel, powder, tablet, or gel capsule form.

[0102] The functional food may also include animal digests, such as any material resulting from chemical and / or enzymatic hydrolysis of clean and undecomposed animal tissue. The functional food may also include dried brewer's yeast, such as a dried inactive agent that is a by-product of the brewing industry. Animal digests and dried brewer's yeast have been found to increase the palatability of functional foods. When included in functional foods, the animal digests constitute about 10% to about 90% of the functional food, and the dried brewer's yeast constitutes about 1% to about 30% of the functional food.

[0103] In some embodiments, the compositions disclosed herein are provided as pharmaceutical or veterinary compositions.In some embodiments, the present disclosure provides compositions comprising the compounds disclosed herein together with at least one pharma- ceutically acceptable carrier, diluent and / or additive (see, for example, Remington: The Science and Practice of Pharmacy, Alfonso R. Gennaro (Editor) Mack Publishing Company, April 1997).As used herein, the term "pharma-ceutically acceptable" refers to a composition, or a combination of agents, materials or compositions, and / or dosage forms thereof, that is within the scope of sound medical judgment, suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reaction or other problem or complication, and that is commensurate with a reasonable benefit / risk ratio. Moreover, the term "pharmaceutical acceptable diluent or carrier" refers to a pharmaceutical acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, that is involved in the carrying or transport of a peptide from one organ or part of the body to another organ or part of the body.

[0104] In some embodiments, the composition comprises at least 10% by weight of the compound disclosed herein.Preferably, the composition comprises at least 10% by weight of DPD.In some embodiments, the composition comprises at least 40% by weight, preferably at least 50% by weight of the compound disclosed herein.In some embodiments, the composition comprises at least 60% by weight, preferably at least 70% by weight of the compound disclosed herein.In some embodiments, the compound disclosed herein is the only active ingredient of the composition.

[0105] In some embodiments, the composition does not include one or more of the following compounds: iso-amyl alcohol, dimethylthiophene, 2-undecanone, tridecane, 2-hexyl-5-methyl 3(2H)-furanone, 2-tridecanone, methyl palmitate, ethyl palmitate, methyl linoleate, and ethyl oleate. More than 10% by weight, preferably more than 1% by weight, more preferably more than 0.1% by weight of isoamyl alcohol; greater than 10% by weight, preferably greater than 1% by weight, more preferably greater than 0.1% by weight of dimethylthiophene; greater than 10% by weight, preferably greater than 1% by weight, more preferably greater than 0.1% by weight of 2-undecanone, greater than 10% by weight, preferably greater than 1% by weight, more preferably greater than 0.1% by weight of tridecane, More than 10% by weight, preferably more than 1% by weight, more preferably more than 0.1% by weight, of 2-hexyl-5-methyl-3(2H)-furanone; greater than 10% by weight, preferably greater than 1% by weight, more preferably greater than 0.1% by weight of 2-tridecanone, More than 10% by weight, preferably more than 1% by weight, more preferably more than 0.1% by weight, of methyl palmitate; More than 10% by weight, preferably more than 1% by weight, more preferably more than 0.3% by weight, preferably more than 0.1% by weight of ethyl palmitate; More than 10% by weight, preferably more than 1% by weight, more preferably more than 0.1% by weight of methyl linoleate; More than 10% by weight, preferably more than 1% by weight, more preferably more than 0.1% by weight of ethyl oleate Does not include.

[0106] The composition may be administered by any suitable route and mode. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired results. The composition may be formulated according to routine procedures for administration by any route, such as parenteral or enteral. Preferably, the composition is administered orally.

[0107] In some aspects, oral administration includes administering the composition in combination with the animal's food, water, or medicine. In some embodiments, oral administration includes applying the composition to a part of the animal's body in a gel or viscous solution or spraying the composition, where the animal ingests the composition by licking. In some embodiments, when the mammal is a ruminant, the composition is injected into the rumen.

[0108] The composition can be in any suitable form, such as liquid, semi-solid and solid dosage forms. The composition can be in the form of tablets, capsules, powders, granules, lozenges, liquids. Preferably, the composition is suitable for oral administration. Such oral compositions include tablets or bolus formulations. As used herein, bolus refers to a single dose of material that can be swallowed. In some embodiments, the composition is an intraruminal bolus.

[0109] In an exemplary embodiment, the composition is a gel capsule comprising the compound disclosed herein. Suitable gel capsules are known in the art and include bovine, porcine and piscine gelatin capsules. Preferably, the gel capsule comprises at least 50 grams of DPD, preferably 50 to 200 grams of DPD.

[0110] In some embodiments, the treatment disclosed herein (administration of a compound disclosed herein) can be combined with another therapy, for example, dry cow therapy.

[0111] For example, the therapy may be combined with abrupt or gradual cessation of milk expression. Abrupt cessation of milk expression refers to stopping the active removal of milk by pumping, milking, or natural feeding (e.g., breastfeeding). Gradual cessation of milk expression refers to a reduction in the amount of milk expressed. This refers to a reduction in the frequency or amount of milk expression.

[0112] In an exemplary embodiment, a lactating dairy animal is administered a compound disclosed herein within 24 hours, preferably within 12 hours, of a "dry-off day". In another exemplary embodiment, a lactating animal, preferably a dairy animal, cow or goat, is provided with a compound disclosed herein 12-24 hours prior to a "dry-off day". In such an embodiment, the animal is no longer milked beginning with the dry-off day until the calf is born (and a new lactation cycle begins). In some embodiments, the compound or composition is administered between 4-8 hours after the last milking. The animal is then no longer milked until after parturition.

[0113] In another example, the therapy may be combined with stepwise feeding. Stepwise feeding is a method to reduce milk production by reducing glucose transport rate to mammary gland. Stepwise feeding can be carried out by many methods known to those skilled in the art. For example, removing concentrate for 14 days before drying off, reducing dry matter intake for 14 days, feeding low energy feed 7 days before drying off, removing hay for 5 days before drying off, etc. (For a review of drying off methods and stepwise feeding, see https: / / edis.ifas.ufl.edu / pdf / AN / AN36000.pdf). For example, the average dry matter intake of a cow during the dry period is approximately 21 kg / day, but this amount may be halved when stepwise feeding is carried out.

[0114] In some embodiments, the use of the compounds disclosed herein may be combined with one or more other treatments used to reduce milk production, prevent or treat mastitis or other infections, or prevent or treat inflammation. Such combinations may be formulated as a single composition, thus containing two or more active ingredients, or the combinations may be administered separately. For example, DPD may be administered several days before or after the administration of one or more other formulations.

[0115] In some embodiments, combination therapy is provided that includes a compound disclosed herein and a prolactin inhibitor. Prolactin, or lactotropin, is an essential protein hormone best known for its role in enabling mammals to produce milk. Inhibition of the formation or release of prolactin is effected by administration of a prolactin inhibitor, resulting in inhibition of milk formation. Examples of prolactin inhibitors include quinagolide and cabergoline.

[0116] Cabergoline is an ergot derivative that inhibits prolactin release. In one study, the overall result of cabergoline administration was reduced prolactin secretion, udder engorgement and milk leakage (in ruminants), and improved lying time. A single injection of cabergoline (5.6 mg) at the dry off reduced prolactin up to 8 days after drying off, resulting in a 28% milk reduction in goats and 22% in dairy cows.

[0117] Cabergoline is an ergoline derivative that stimulates dopamine D2 receptors in lactotroph cells of the pituitary gland, thus inhibiting prolactin release. A single intramuscular injection of cabergoline is sufficient to reduce plasma prolactin concentrations during the first week of dry-off compared to an untreated placebo group. Furthermore, a decrease in lactose concentration and an increase in lactoferrin concentration in milk were detected (Boutinaud et al, 2016. Cabergoline inhibits prolactin secretion and accelerates involution in dairy cows after dry-off. Journal of dairy science, 99(7), 5707-5718). Cabergoline for intramuscular administration was marketed under the trade name Velactis. Treatment with Velactis resulted in a lower incidence of lactitis after the 7th day after calving compared to the control group, 20.5% and 26%, respectively. Milk leakage was also lower compared to the control group, 2% and 11%, respectively. Moreover, behavioral observations showed that administration of Velactis resulted in less udder pain (in ruminants) compared to the control group. However, following reports of adverse events, the drug is no longer approved for use in the European Union. 319 dairy cows had serious adverse events after treatment, such as recumbency (208) and deaths (71) (European Medicines Agency, 2016).

[0118] Another prolactin inhibitor is quinagolide: it is a dopamine receptor agonist. It stimulates dopamine D2 receptors and inhibits the release of prolactin from the anterior pituitary gland. A group of cows in early lactation were given daily intramuscular injections of 1 mg of quinagolide for 9 weeks. This reduced milking-induced prolactin release but not basal prolactin concentrations. Moreover, milk production was found to be reduced by 5.3 kg / day during the last 4 weeks of treatment compared to the placebo group (Lacasse et al.,2011.Effect of the prolactin-release inhibitor quinagolide on lactating dairy cows.Journal of dairy science,94(3),1302-1309). Another group of cows were given intramuscular injections of 4 mg twice daily from 5 days before dry-off to 13 days after dry-off. During this treatment, prolactin concentrations in blood, milk and mammary secretions were reduced. In addition, milk production was found to be reduced by 18 kg / day and 25 kg / day, respectively, compared to the placebo group. In this experimental design, another group of cows was fed only hay instead of the normal lactation diet, and a reduction in prolactin levels in the blood and milk was also found. Milk production was reduced by 10 kg / day. The difference between cows treated with quinagolide and the group fed only hay was in the metabolites in the blood. The first group showed an increase in blood glucose levels, while the levels of other metabolites were not affected. In the second group, a decrease in blood glucose levels, a decrease in most amino acids, and an increase in blood levels of beta-hydroxybutyrate and non-esterified fatty acids were observed.Dietary restriction resulted in a large decrease in milk production, but quinagolide may be a good alternative to reduce milk production without interfering with metabolism (Ollier, Zhao and Lacasse, 2014. Effects of feed restriction and prolactin-release inhibition at drying off on metabolism and mammary gland involution in cows. Journal of dairy science, 97(8), 4942-4954). The drawback of using quinagolide is the frequency of administration. Because of the additional cost, dairymen may choose to administer prolactin inhibitors only to cows with daily dry milk yields of more than 15-18 kg (33-40 lbs).

[0119] The acidogenic mineral bolus contains anionic salts and induces a temporary metabolic acidosis. One study showed that cows receiving two 196 g boluses on the second day after treatment lost 2.6 kg / day of milk compared with a loss of 1.2 kg / day in cows receiving a single bolus and a loss of 0.23 kg / day in the control group. Another study showed that cows receiving two boluses lost feed intake during the first three days after treatment. The mineral composition of the oral boluses (196 g each) was 10.4% NH4Cl, 51.9% calcium chloride, 20.8% calcium sulfate, 12.6% water, and 4.3% coating agent (mono- and diglycerides of fatty acids esterified with acetic acid). Each bolus contained approximately 20 g of NH4Cl (about 10.4% of the total bolus weight). Milk production was reduced on the second and third days after treatment. Furthermore, the pH of urine from treated cows was lower than that of control cows. Furthermore, udder pressure was lower in cows treated with two boluses compared to the control group, but the incidence of milk leakage did not differ between treated and control groups. Therefore, the ingress of microorganisms that may cause mastitis may still occur. Maynou et al. (2018, Effects of oral administration of acidogenic boluses at dry-off on performance and behaviour of dairy cattle. Journal of dairy science, 101(12), 11342-11353) provide two possible explanations for the reduced feed intake during the first three days after treatment. The first explanation is that it is due to a change in the acid-base status of the cow's rumen. The second explanation is that it may occur due to a potential damage to the rumen wall. The reduced milk yield is partly due to the reduced feed intake. Beta-hydroxybutyrate concentrations were lower compared to the control group.This was explained by reduced feed intake, which in turn resulted in reduced levels of metabolized butyrate in the rumen wall (Maynou et al., 2018).

[0120] Another composition that promotes dry-off is casein hydrolysate (CNH), which induces loss of integrity of tight junctions in the mammary gland (Shamay et al., 2003. Infusions of casein hydrolysate into the mammary gland disrupt tight junction integrity and induce involution in cows. Journal of Dairy Science, 86(4), 1250-1258; Ponchon et al., 2014. Effects of intramammary infusions of casein hydrolysate, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, and lactose at drying-off on mammary gland involution. Journal of dairy science, 97(2), 779-788). Shamay et al. demonstrated that infusion of CNH is an effective and gentle method to reduce milk production during lactation and to dry chronically infected udders and treatment-resistant single udder quarters.

[0121] Seeth et al. (2016. Drying-off single udder quarters of dairy cattle during lactation using casein hydrolysate. Milk Science International 69:23-26) demonstrated that casein hydrolysate infusions were an effective and gentle method to down-regulate milk production during lactation and dry out chronically infected udders and treatment-resistant single udder quarters when six intracisternal infusions of casein hydrolysate were administered within three treatment days into each treated udder quarter. In a pH paper by Justine Elena Britten (2019) (Evaluation of Casein Hydrolysate as an Alternative Dry-Off. Treatment and Milk Quality Management Tool in Dairy Cows Utah State University. Dissertations Graduate Studies 5-2019), casein hydrolysate administration was evaluated and concluded to be a promising tool for dry-off. Animals treated with casein hydrolysate did not show any signs of discomfort or pain. These studies showed that intramammary infusion of casein hydrolysate is safe for dairy cows, shows some efficacy against mastitis, and may have added value in reducing mastitis in lactating and dry cows. This was confirmed by Britten et al. (2021) (Comparison of Bovine Mammary Involution and Intramammary Infections Following Intramammary Treatment with Casein Hydrolysate and Other Conventional Treatments at Dry-Off. Animals 2021,11(8),2360).

[0122] In some embodiments, a combination therapy is provided that includes a compound disclosed herein and an anti-inflammatory agent. Such agents can be administered to suppress inflammatory responses and reduce tissue damage, such as tissue damage in the milk gland during dry off. Anti-inflammatory agents include, for example, nonsteroidal anti-inflammatory agents (cox / lox inhibitors), such as ibuprofen, paracetamol, aspirin, diclofenac, ketoprofen, tolmetin, etodolac, and fenoprofen. Natural anti-inflammatory agents, such as curcumin, ginger, spirulina, cayenne, cinnamon, clove, sage, rosemary, black pepper, natural aspirin, boswellia, sanguinaria, and green tea, may also be used.

[0123] In another example, it may be used in conjunction with teat sealants. After dry cow therapy, cows are often given internal teat sealants. Internal teat sealants typically involve a paste injected into each teat, which creates a physical barrier against microorganisms. Teat sealants reduce new infections for several days after drying off, when the natural barrier, the keratin plug, made of a waxy substance at the end of the teat, has not fully formed.

[0124] Teat sealants may be administered internally or topically. Antibiotics are commonly used as a treatment that is inserted into the teats before the sealant is applied. This may be followed by post-milking teat dips or sprays. After treatment, cows should spend at least 30 minutes in a clean area and should not be allowed to walk long distances after drying off. Once the cows have been dried off, the condition of the cow's (ruminant) udder is checked regularly for signs of inflammation and infection.

[0125] In some embodiments, the treatments disclosed herein (administration of the compounds disclosed herein) can be combined with antibacterial agents, such as antibiotics or antifungals. Without wishing to be bound by theory, the present disclosure provides that the compounds disclosed herein can prevent intramammary infections, while the antibacterial agents can then exert their effect on remaining infections or help prevent new infections.

[0126] Exemplary antibacterial agents that may be used in combination therapy include antifungals such as miconazole, ketoconazole, econazole, terbinafine, ciclopirox, tolnaftate, sertaconazole, sulconazole, amphotericin b, chloroxylenol, clioquinol, butenafine, naftifine, nystatin, and clotrimazole. Exemplary antibiotics include penicillins, tetracyclines, cephalosporins, quinolones, lincomycins, macrolides, sulfonamides, glycopeptides, aminoglycosides, and carbapenems.

[0127] The current method to prevent the occurrence of intra-mammary mastitis infection, i.e. dry cow therapy, involves the injection of antibiotics and / or teat sealant into the udder quarters (of ruminants) on the dry day with the aim of preventing and treating infection during the dry period. The use of antibiotics reduces existing infections as well as new infections during the early dry period. Dry cow therapy can be divided into "blanket" and "selective" treatments. Blanket dry cow therapy is used for the majority of treatments in the United States (93% of cows) (USDA 2016). In blanket therapy, antibiotics are injected into all quarters of all cows in the herd, regardless of their intramammary infection status. However, the application of blanket dry cow therapy to cows that do not require antibiotics may lead to antimicrobial resistance.

[0128] In contrast, selective dry cow therapy means that antibiotics are given only to cows that have an infection or are at unusually high risk of infection. Candidates for selective dry cow therapy are cows that have low somatic cell counts during the three months prior to drying off. Herds with a low incidence of subclinical mastitis are well suited for selective dry cow therapy.

[0129] Optimized conditions for dry cows are known to those skilled in the art. Stress can have a negative effect on appetite and immunity, so it is important to reduce it as much as possible during the dry period. Social stress can be reduced by avoiding herd turnover and keeping the social hierarchy as close as possible. Some dairymen separate dry cows from the rest of the herd and guarantee that the cows are no longer milked. Also, environmental conditions, such as ventilation and temperature, are important. Improved feed is generally provided during the dry period.

[0130] In a preferred embodiment, treatment with the compounds disclosed herein is combined with compounds of formula I and / or formula II disclosed in International Publication No. WO021 / 182958, the entire contents of which are incorporated herein by reference.

[0131] As used herein, a compound according to Formula I below (International Publication No. WO2021 / 182958) is as follows: [ka] Here, R 1 and R 2 are independently selected from optionally substituted linear or branched alkyl, optionally substituted linear or branched alkenyl, optionally substituted linear or branched alkynyl, optionally substituted aryl, optionally substituted cycloalkyl, and optionally substituted heterocycloalkyl. Preferably, the compound of formula I is propylpropanethiosulfonate (PTSO).

[0132] As used herein, a compound according to Formula II (International Publication No. WO2021 / 182958) is as follows: [ka] Here, R 3 and R 4 is independently selected from optionally substituted linear or branched alkyl, optionally substituted linear or branched alkenyl, optionally substituted linear or branched alkynyl, optionally substituted aryl, optionally substituted cycloalkyl, and optionally substituted heterocycloalkyl. Preferably, the compound according to formula II is propyl propane thiosulfinate (PTS).

[0133] The combination of the compounds of the present invention with compounds according to formula I (International Publication No. WO2021 / 182958) or formula II (International Publication No. WO2021 / 182958) makes it possible to both treat existing infections before drying off and to prevent new infections.

[0134] In some embodiments, the compound of formula I (International Publication No. WO2021 / 182958) and / or the compound of formula II (International Publication No. WO2021 / 182958) is administered before the dry day, preferably one month or two months before the dry day. The compound of the present invention is administered, for example, before or after the dry day, as disclosed herein.

[0135] The present disclosure further provides a kit-of-parts comprising a first composition comprising a compound of formula I (International Publication No. WO2021 / 182958) and / or a compound of formula II (International Publication No. WO2021 / 182958), and a second composition as disclosed herein comprising a compound of the invention.

[0136] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0137] When used in connection with numerical values ​​(approximately 10, about 10), the word "approximately" or "about" preferably means that the value can range from around 10 to 1% of the given value.

[0138] The compounds and compositions disclosed herein are useful as therapeutics and in therapeutic treatments, and therefore may be useful as pharmaceuticals and may be used in methods of preparing pharmaceuticals. In some embodiments, the disclosure provides methods that are not treatments of the human or animal body and / or that do not include a step for modifying the genetic identity of a human germline, where the cells are not human germline cells.

[0139] All patent and journal publications cited herein are hereby incorporated by reference in their entirety.

[0140] The present invention will be further described in the following examples, which are not intended to limit the scope of the invention but are merely intended to illustrate the invention.

[0141] Working Example

[0142] Several methods have been applied and studied for drying off cows. For example, Pattamanont et al. (2020. https: / / edis.ifas.ufl.edu / publication / AN360) provide an overview of most of the methods used, as well as a summary of advantages and disadvantages. Since the likelihood of new intramammary infections occurring is 77% higher compared to lactation, there is a strong need for new compounds, compositions and / or methods, or combinations of existing and new methods, that reduce the risk of new infections during the dry off period. The examples described below demonstrate the effect of a single oral dose of DPD on milk yield.

[0143] Example 1. The effect of 40ml and 80ml doses of DPD (di-n-propyl disulfide) on milk production in cows

[0144] Scope: Various doses of DPD were administered orally to cows and the effect on milk production was measured.

[0145] Research design:

[0146] Fifteen cows from three different farms were used in study 1 and 30 cows from four different farms in study 2. The start of the dry period was 40-60 days before calving for all cows. The cows were milked twice a day at a fixed time, and cows producing more than 11.5 liters of milk per day were selected 24 hours before the DPD treatment. No special measures were taken to promote dryness, such as changes in feed allocation or milking frequency. The cows were milked twice a day. The cows were housed in free stables. Pork gelatin capsules with DPD or placebo gel capsules with water were administered 7-14 days before the start of the dry period. Dairymen were not informed which cows were treated.

[0147] For study 1, gel capsules containing 40 ml of DPD were administered orally via a bolus shooter, while the placebo group gel capsules contained tap water as previously described. The placebo group had 8 cows, whereas the treatment group had 7 cows.

[0148] For Study 2, 80ml of DPD was placed in a gelatin capsule and tap water was placed in a gelatin capsule for the placebo group. Nine cows were given a single dose of DPD and nine cows were given a single dose of the placebo capsule 7-14 days before the start of the dry period. The cows were randomly assigned to the DPD or placebo group. This was done for all farms with groups that were dried off at the same time so that every farm had 50% of the cows in the treatment group and 50% in the control group.

[0149] The capsules were administered between the morning and afternoon milkings. On day t = 13, the DPD-containing capsules and the placebo capsules were administered.

[0150] The following hypotheses were tested and compared by statistical analysis: H0 was defined as no difference between the relative milk production before and after treatment between the DPD bolus and placebo groups, and H1 was defined as a difference between the relative milk production before and after treatment between the DPD and placebo groups.

[0151] Results and Discussion

[0152] Description of cows

[0153] Of the treatment group, the 15 cows had a mean age of 5.5 years (standard deviation 2.1 years), a mean calving age of 2.9 years (standard deviation 1.9 years), and a mean milk yield of 20.2 liters per 24 hours (standard deviation of 4.5 liters was calculated).

[0154] Of the placebo group, the nine cows had a mean age of 4.5 years (standard deviation 1.3 years), a mean calving age of 2.8 years (standard deviation 1.2 years), and a mean milk yield of 23 liters per 24 hours (standard deviation 4 L), which was determined 24 hours before treatment.

[0155] Study 1. Milk yield of cows treated with 40ml of DPD

[0156] The milk yield of the cows showed a large variation from day to day. This is normal since cows have a high variability in milk yield. To demonstrate the day to day variation in milk yield, the milk production of four cows per treatment is shown in Table 1 below. For this study, a total of eight cows were used, therefore not all data are shown in this table.

[0157] [Table 1]

[0158] In Figure 1 the average milk production of 8 placebo cows is shown. The absolute milk yield per cow may differ significantly, but a clear downward trend was observed after treatment. The administration of capsules containing water did not show a rapid change in the steady decline of milk yield (t=13 days). ND=not determined.

[0159] Data Processing

[0160] The data were processed as follows: based on the data obtained from days 0, 1, 2, 3...12, the expected milk yield on days 13, 14, 15...20 was calculated for each cow by linear regression. On day t=13, a placebo capsule or a capsule containing DPD was administered intraruminally. The expected data from day t=13 (when no administration of placebo or capsule containing DPD was performed) was compared with the actual milk yield after administration, and the milk drop was calculated. This was done for all cows in the study.

[0161] In the following, the mean milk yield reduction is calculated as the difference between the observed daily milk yield and the expected daily milk yield, which is calculated by comparing the expected milk yield obtained via linear regression with the actual milk yield obtained after treatment with placebo or DPD.

[0162] In Table 2 below the results for the placebo treated cows are shown.

[0163] [Table 2]

[0164] Table 2. Mean milk yield reduction (difference) for cows treated with placebo. "Difference" refers to the mean observed actual milk yield minus the predicted milk yield, obtained by linear regression.

[0165] This approach is also applied to a group of cows treated with 40 ml capsules and a group of cows treated with 80 DPD. In Table 3 this approach is shown for 40 ml DPD.

[0166] [Table 3]

[0167] Table 3. Mean milk yield reduction (difference) in cows treated with 40 ml of DPD.

[0168] Then, the difference between the treatment and the placebo was tested to see if it was significantly different. The null hypothesis was that the difference was zero. Using a permutation test (5000 samples), the P-value was calculated for these differences. The results are shown in Table 4 below.

[0169] [Table 4]

[0170] Table 4. Significance of the effect on milk yield of treatment with 40 ml DPD compared to cows treated with placebo. Mean differences are expressed in litres of milk per cow per day. "2.5% percentile" and "97.5% percentile" bound the confidence interval for the null hypothesis.

[0171] The P value reveals that treatment with 40 ml of DPD did not result in a significant decrease in milk yield. However, two cows had a decrease in milk yield after treatment, while the other two cows did not. It was observed that the milk yield decrease was more variable at the low DPD doses than at the high DPD doses. Upon closer observation, the work procedures during the low dose experiments were less structured in terms of the moment of the day when milking occurred, and there was little supervision during the conduct of these experiments. This resulted in a relatively high standard deviation, which made the effect of low dose DPD on milk yield decrease less clear. Therefore, in Example 2, the dose-response curve of DPD dose and milk yield after administration was constructed under standardized and closely monitored experimental conditions. Therefore, the results for Example 2 are considered to be close to reality.

[0172] Study 2. Milk yield of cows treated with 80ml of DPD

[0173] A similar experiment to that described above with 40 ml of DPD administered was performed using 80 ml of DPD instead, and the results are shown in Table 5 below.

[0174] [Table 5]

[0175] Table 5. Mean milk yield reduction (difference) in cows treated with 80 ml of DPD.

[0176] It was then tested whether the differences between the 80 ml treatment and the placebo were significantly different. The null hypothesis defines the difference between the treated and placebo cows as zero. P values ​​were calculated for these differences using a permutation test (5000 samples). The results are shown in Table 6 below.

[0177] [Table 6]

[0178] Table 6. Significance of the effect on milk yield of treatment with 80 ml of DPD compared to cows treated with placebo. Mean differences are expressed in litres of milk per cow per day. "Percentile 2.5%" and "Percentile 97.5%" bound the confidence interval for the null hypothesis.

[0179] The P values ​​reveal that treatment with 80 ml of DPD did not result in a significant decrease in milk production.

[0180] Conclusion and discussion

[0181] No significant differences were found between the relative milk production of the DPD group before treatment and the placebo group. A significant reduction in cows treated with 80ml of DPD compared to the placebo group was seen over the next three days after treatment (average 6 litres per cow per day), with milk production recovering after this period.

[0182] This procedure in combination with other drying techniques, such as abrupt drying, graduated milking, graduated feeding, or other methods, is expected to reduce milk production and reduce the risk of new (ruminant) udder infections and stress / soreness.

[0183] Rajala-Schultsz et al. (2005. Association between milk yield at dry-off and probability of intramammary infections at calving. Journal of Dairy Science, 88(2), 577-579) found that for every 5 kg increase in dry-off milk yield above 12.5 kg, the probability of environmental intramammary infection at calving increases by 77%. Treating cows with DPD therefore results in a highly significant reduction in the probability of environmental IMI at calving. This was supported by Odensten et al. (2007, Metabolism and udder health at dry-off in cows of different breeds and production levels. Journal of Dairy Science, 90(3), 1417-1428), who found that cows producing more than 11.5 kg of milk per day in the week before drying off had a higher percentage of open teat canals in the second (30%) and third (12%) weeks of the dry period and were more likely to develop IMI shortly after calving (21.8%) compared to cows producing less milk. Treatment with DPD would be expected to result in a lower percentage of open teat canals and a lower probability of IMI shortly after calving when the cow produces less than 11.5 kg per day. A study by Maynou et al. (2018) found that milk production was reduced in the group receiving two boluses of acidogenic mineral bolus on the second and third days after treatment, with a reduction of 2.6 liters of milk production on the second day compared to a reduction of 0.23 liters for the placebo group. A reduction of 7.7 liters of milk production was observed on the second day after treatment compared to a reduction of 0.75 liters of milk production per day for the placebo group, with the DPD group demonstrating a greater effect compared to the product. This indicates that DPD is a more powerful dry milk product than acidogenic mineral bolus.In a study by Ollier, Zhao, and Lacasse (2014), quinagolide treatment reduced milk production by 7 liters compared to the placebo group. The advantage of DPD compared to quinagolide is the ease of administration: quinagolide must be injected frequently, whereas DPD is administered orally and a single dose is sufficient.

[0184] Example 2. Dose-Response DPD Treatment and Milk Yield

[0185] Range. In this example, the correlation between milk yield reduction and various doses of synthetic DPD was determined.

[0186] Study design. Twenty-seven cows (Holstein Friesians) from two farms were used. The start of the dry period was 40-60 days before calving for all cows. The cows were milked twice daily at a fixed time, and cows with a milk production of more than 12.5 liters per day were selected 24 hours before DPD administration. No special measures were taken to induce dryness, such as changes in feed allocation or milking frequency. The cows were milked twice daily. The cows were housed in free stables. Pork gelatin capsules with DPD or placebo gel capsules with water were administered 7-14 days before the start of the dry period.

[0187] For this study, various doses of synthetic DPD (>98% purity) were administered to cows producing at least 12.5 kg of milk per day. The following doses were applied: 0, 10, 20, 50, 100, 150, 175, 200 ml of pure DPD. For each dose, three cows were used.

[0188] Milk production was monitored for 14 days before the various doses were administered. The gel capsules were administered orally by bolus shooter, and the gel capsules for the placebo group were filled with tap water. The capsules were administered between the morning and afternoon milkings. On t=14, DPD-containing and placebo capsules were administered, and milk production was monitored for 5 days.

[0189] Results. In table 7, milk yield and change in milk yield (%) after administration of various doses of DPD are presented. A positive value of "% decrease" indicates a decrease in milk yield, while a negative value indicates an increase in milk yield. The reference treatment, in which a capsule of water was administered instead of a capsule with DPD, did not show any effect on milk yield in the following 5 days. However, a dose of 20 ml of DPD showed a clear decrease on milk yield compared to the milk yield before treatment, and the decrease effect became stronger when more DPD was administered. No further decrease in milk yield was observed when more than 80 ml was administered. Moreover, the cows showed a relaxed and quiet behavior and no change was observed in feeding behavior. However, the cows seemed to become more active when more than 100 ml was administered.

[0190] [Table 7]

[0191] Discussion and conclusion. This study showed that a dose-response curve could be constructed between the dose of DPD and the reduction in milk yield. The reduction in milk yield was evident when 20 ml or more of DPD was administered. The maximum response was obtained after 80 ml or more of DPD was administered. Up to 80 ml of DPD, no undesirable behavior of the cows was observed, but when 80 ml or more of DPD was administered, the cows became restless and showed a more active behavior. The effect of DPD disappeared 3-4 days after treatment, which strongly indicates that the treatment does not result in harm to the health of the cows ((ruminant) udder). In contrast, DPD treatment opens the possibility of preventing or reducing milk leakage after a sudden dry-off and thus preventing new mastitis infections. Secondly, the treatment also reduces or prevents the painful udder pressure associated with stress. Therefore, administering DPD to cows during a sudden dry-off is a valuable tool for the health of the cows and the profitability of the dairy farm.

[0192] Example 3. Effect of synthetic (>98% purity) DPD on udder pressure, stress behavior and milk leakage in highly productive cows after a rapid dry off

[0193] List of abbreviations: IMI (Intramammary Infections): Intramammary infection; sDCT (selective dry cow therapy): Selective dry cow therapy; bDCT (blanked Dry Cow Therapy): Blank dry cow therapy; ITS (Intramammary Teat Sealer): Intramammary teat sealer; DIL (Days in Lactation): (Number of lactation days); SCC (Somatic cell count): (Somatic cell count).

[0194] Scope: Especially in high lactating cows, sudden dry off is a target for new infections due to stress factors such as discomfort due to high udder pressure resulting in stress or milk leakage. New IMI (intramammary infection) is mostly caused by bacteria entering the udder through the teat canal. Different doses of DPD were orally administered to cows and the effect on discomfort and udder leakage was measured.

[0195] research design

[0196] Holstein-Friesian dairy cows producing at least 12.5 kg of milk per day in their last lactation were selected. These cows were treated immediately after their last milking before being suddenly dried off with 80 ml of di-n-propyl disulfide (DPD) in the three studies of this example. Due to the limited number of cows available, a control group was only available in study 3. Treated cows were monitored for udder pressure, milk leakage, and stress every 12 hours after their last milking for the first 72 hours.

[0197] Animals and Design

[0198] 46 Holstein-Friesian dairy cows from seven farms in the Netherlands were enrolled in a two-month field study. The housing conditions and cow information are presented in Table 8 below.

[0199] [Table 8]

[0200] After the last milking, the cows were isolated from other lactating cows. Only on farms 2 and 6 could the cows hear the milking machine. After isolation, the cows were administered 80 ml of synthetic DPD. The cows were then monitored by the dairy farmers for 72 hours after a quick dry-off and scored every 12 hours. The dairy farmers visually scored the cows for milk leakage (scored yes / no), udder pressure and stress according to a standard scoring form (see Tables 9 and 10 below). The cows' somatic cell counts were extracted from the last milk recorded before the dry-off and the first milk recorded after calving (5-30 days of lactation).

[0201] [Table 9]

[0202] [Table 10]

[0203] Data analysis: The control group was scored for milk leakage, udder stress (ruminants) and stress and compared with data previously presented in the literature. For milk leakage, the data were compared assuming that 24.5% of cows leak milk after drying off (De Prado-Taranilla et al.,2020. Incidence of milk leakage after dry-off in European dairy herds,related risk factors,and its role in new intramammary infections. Journal of Dairy Science,103(10),9224-9237.). Udder pressure (Bertulat et al., 2013. Measurement of fecal glucocorticoid metabolites and evaluation of udder characteristics to estimate stress after sudden dry-off in dairy cows with different milk yields. Journal of Dairy Science, 96(6), 3774-3787) and stress (Chapinal et al., 2014. Changes in lying behavior after abrupt cessation of milking and regrouping at dry-off in free stall-housed cows: A case study. Journal of Veterinary Behavior, 9(6), 364-369) were converted into a score sheet with expected outcomes (Table 11 below).

[0204] [Table 11]

[0205] result

[0206] 46 Holstein-Friesian cows showed an average milk production of 22.3 kg / day on the last day before drying off (milk yields varied from 15.6 to 29.7 kg / day). No side effects were observed in any of the cows during the study. The results of milk leakage, udder pressure and stress are reported in Figures 2, 3 and 4.

[0207] The average SCC of the 46 cows at the last milk recording of the last lactation was 89,000 cells / ml (SCC ranged from 45,000 to 106,000 cells / ml) and the somatic cell count at the first milk recording of the new lactation was 58,000 cells / ml (range 36,000 to 112,000 cells / ml). None of the cows had milk fever at calving.

[0208] Discussion

[0209] The milk drop demonstrated by administration of 80 ml of pure DPD was strong enough to successfully dry off high-producing cows. Previous studies have shown that high milk production on the last day before drying off results in udder pressure and milk leakage (Chapinal et al., 2014; Bertolat et al., 2013; De Prado-Taranilla et al., 2020), and these observations were consistent with those shown in the experimental controls as presented in this example. In this study, it was also demonstrated that administration of the indicated doses of synthetic DPD resulted in lower udder pressure, lower stress, and less milk leakage during the sudden dry off compared to the control group.

[0210] The purpose of applying DPD at the onset of the dry-off is to reduce the risk of new infections through the udder teats. Milk leakage, mainly stimulated by high udder pressure, is a common cause of new IMIs during the onset of the dry-off period (Klaas et al.,2005.Cow-Related Risk Factors for Milk Leakage.Journal of Dairy Science,88(1),128-136; Rajala-Schultz et al.,2005.Short Communication: Association Between Milk Yield at Dry-Off and Probability of Intramammary Infections at Calving.Journal of Dairy Science,88(2),577-579; Gott et al.,2016.Intramammary infections and milk leakage following gradual or abrupt cessation of milking.Journal of Dairy Science,99(5),4005-4017). However, despite the reduced udder pressure and ITS resulting from the treatment, the percentage of cows with milk leakage remained low, which nevertheless resulted in a low incidence of IMI (Example 4).

[0211] The dry period is the period of highest antibiotic use in the dairy sector, especially to prevent new IMI or treat subclinical mastitis (Kuiper et al., 2014. Antibiotic use in dairy herds in the Netherlands from 2005 to 2012. Sciencedirect.com. https: / / www.sciencedirect.com / science / article / pii / S0022030215009054?ref=pdf_download&fr=RR-2&rr=738fe0be1d72b7d3). DPD is effective in reducing new infections during the dry period (as demonstrated in Example 4) and can reduce the need for long-acting antibiotics during the dry period and after calving.

[0212] conclusion

[0213] DPD may be a useful additive to support cows after a sudden dry off. DPD treated cows had significantly lower udder pressures compared to control cows. This correlated with a lower percentage of cows with milk leakage and lower stress levels, resulting in fewer new IMIs. This is a valuable positive contribution to farm economics and cow wellbeing.

[0214] Example 4 - Effect of DPD-rich onion extract on udder pressure, stress behavior and milk leakage in highly productive cows after sudden drying off

[0215] Scope: In this example onion extract was enriched with 80ml DPD to obtain DPD enriched onion extract (=DPD enriched OE). Two studies were performed after a single administration of 150ml DPD enriched OE. After administration of 150ml DPD enriched OE an acute dry off was performed and the effect on three dry off variables was investigated: milk leakage (study 1), stress behaviour and udder pressure (study 2).

[0216] Study 1. Effects on milk leakage and stress levels.

[0217] Study design. Cow description: Thirty-one dairy cows (Holstein-Friesian) from six farms in the Netherlands were enrolled in this field study. The start of the dry period was 40-60 days before calving for all cows, which corresponds to the optimal dry period interval (Capuco et al., 1997. Mammary Growth in Holstein Cows During the Dry Period: Quantification of Nucleic Acids and Histology. Journal of Dairy Science, 80(3), 477-487). The conditions of care and cow information are presented in Table 12 below.

[0218] [Table 12]

[0219] The cows had a minimum milk production of 12.5 kg per day on the dry day. The cows were dried off suddenly and transferred directly to the dry group after the final milking.

[0220] After the last milking, the cows were treated with ITS and then with 150 ml of OE enriched with DPD. All cows were housed in free stalls separate from the milking cows.

[0221] Cows were followed by the dairy farmer for 72 hours and scored every 12 hours after the quick dry off. The dairy farmer had to visually score milk leakage and assign a qualitative yes / no score, udder pressure and stress according to a standard scoring form (see Tables 10 and 11 in Example 3). Cows' somatic cell counts were taken from the last milk recorded before the dry off and the first milk recorded after calving (5-30 days of lactation).

[0222] Data processing: Due to the small number of cows, data for the control group were obtained from the literature: udder pressure and stress scores were compared with expected data as previously described (Chapinal et al., 2014. Changes in lying behavior after abrupt cessation of milking and regrouping at dry-off in freestall-housed cows: A case study. Journal of Veterinary Behavior, 9(6), 364-369; Bertulat et al., 2013. Measurement of fecal glucocorticoid metabolites and evaluation of udder characteristics to estimate stress after sudden dry-off in dairy cows with different milk yields. Journal of Dairy Science, 96(6), 3774-3787). In example 3, this was demonstrated to be a reliable approach. Regarding milk leakage, the data was compared with previous studies which demonstrated that 24.5% of cows experience milk leakage after dry-off (De Prado-Taranilla et al., 2020. Incidence of milk leakage after dry-off in European dairy herds, related risk factors, and its role in new intramammary infections. Journal of Dairy Science, 103(10), 9224-9237).

[0223] H0: After administration of DPD-enriched OE (containing 80% natural DPD) at the beginning of the dry off period, cows score equally or better in terms of reduced udder pressure, reduced stress levels and reduced milk leakage. As there is no control group in this experiment, no statistical analysis was performed. The data obtained has been compared with previous data obtained from the literature.

[0224] The following study was conducted to analyze whether 150ml of DPD-enriched OE was effective in preventing sudden dry off stress, udder soreness (in ruminants), and milk leakage in high producing cows (>12.5 kg / day).

[0225] Study 2 - Udder pressure after sudden dry off

[0226] Study design. Animal description: Six dairy cows (Holstein-Friesian) were enrolled in this field study on one farm in the Netherlands during July 2022. The start of the dry period was 40-60 days before calving for all cows, as this is the optimal interval before the dry period begins. The cows had a minimum milk production of 12.5 kg per day on the dry day. The cows were dried off suddenly and transferred directly to a dry group where they could not hear the milk robot after the final milking. After the final milking, every second cow (2, 4, 6) was treated with ITS and then with 150 ml of OE enriched with DPD. The other three cows (control group) were only treated with ITS after the final milking. All cows were housed in free stalls.

[0227] All cows were followed for 72 hours after drying off and udder pressure was measured by a penetrometer (Medista 5000 Digital firmness instrument supplied by Medista, 13 Rue du Bastringue 76440 Serqueux, La France). Cows were measured directly before and after the last milking (maximum and minimum pressures were estimated, respectively). In addition, udder pressure was monitored 12, 24, 36, 48, 60 and 72 hours after the last milking (Table 13 below). All cows were measured with a penetrometer on the left front tuft, 10 cm directly above the teat (the location marked with a red marker during the first measurement).

[0228] [Table 13]

[0229] H0: Cows treated with DPD-enriched OE showed lower udder pressure after sudden dry-off than untreated cows after dry-off. Results were statistically analyzed by t-test for difference in means.

[0230] result

[0231] Research 1

[0232] Thirty-one Holstein-Friesian cows had an average milk yield of 24.5 kg / day on the last day before drying off (milk yields ranged from 17.7 to 29.9 kg / day). No side effects were observed in any of the cows during the study. The results for milk leakage, udder pressure and stress are shown in Figures 5, 6 and 7.

[0233] The average SCC of the 46 cows at the last milk recording of the last lactation was 84,000 cells / ml (SCC varied from 31,000 to 97,000 cells / ml) and the somatic cell count at the first milk recording of the new lactation was 61,000 cells / ml (SCC varied from 36,000 to 97,000 cells / ml). None of the cows had milk fever at calving.

[0234] Research 2

[0235] Three test cows and three control cows were measured for their udder pressure after a sudden dry off. Two of the three control cows showed milk leakage after 36 hours, whereas none of the test cows treated with DPD-enriched OE showed milk leakage. The udder pressure results are reported in Figure 8.

[0236] FIG. 8 demonstrates that mammary pressure was significantly reduced following administration of 150 ml of DPD-enriched OE.

[0237] Discussion and conclusion: In this example, it is clearly demonstrated that a single dose of OE enriched with DPD results in lower udder pressure, less milk leakage and less stress behavior. In the literature, a positive relationship is shown between milk leakage after abrupt drying and the occurrence of mastitis. Milk leakage is the result of teat canal opening after abrupt drying, therefore a significant number of mastitis cases are prevented, which is economically important. Moreover, this method is very beneficial for the well-being of the cows, since abrupt drying is a painful process, resulting in high udder pressure and many stress behaviors.

[0238] Example 5. Dose-Response PTSO Treatment and Milk Yield

[0239] Range. In this example, it is shown that di-n-propylthiosulfonate (PTSO), a compound chemically related to DPD, exhibited the same or similar effect on milk yield reduction as DPD. In this example, PTSO was used as the demonstrative compound. Furthermore, the correlation between milk yield reduction and various doses of PTSO was determined.

[0240] Study design. Twelve cows (Holstein-Friesian) from one farm were used. The start of the dry period occurred 40-60 days before calving for all cows. The cows were milked twice daily at a fixed time, and cows producing more than 12.5 liters of milk per day 24 hours before PTSO administration were selected. No special measures were taken to induce dry period, such as changes in feed allocation or milking frequency. The cows were milked twice daily. The cows were housed in free stables. Pork gelatin capsules with PTSO or placebo gel capsules with water were administered 7-14 days before the start of the dry period.

[0241] For this study, various amounts of synthetic PTSO (>98% purity) were administered to cows with milk yields of at least 12.5 kg per day. The following doses were applied: 0, 10, 20, 40 ml of pure PTSO. For each dose, three cows were used.

[0242] Milk production was monitored for 14 days before the various doses were administered. The gel capsules were administered orally by bolus shooter, and the gel capsules for the placebo group were filled with tap water. The capsules were administered between the morning and afternoon milkings. On t=14, PTSO-containing and placebo capsules were administered, and milk production was monitored over 48 hours.

[0243] Results. In Table 1, milk yield and percent change in milk yield after administration of various doses of PTSO are presented. Positive values ​​of "% decrease" indicate a decrease in milk yield, while negative values ​​indicate an increase in milk yield. Reference treatment, during which a capsule of water was administered instead of PTSO, showed no effect after 24 and 48 hours. However, administration of 20 ml of PTSO obviously decreased milk yield compared to the milk yield before treatment, and the decreasing effect was stronger when more PTSO was administered.

[0244] [Table 14]

[0245] Discussion and conclusion: This study showed that there was a dose-response curve between the dose of PTSO and the reduction in milk yield. The effect was evident when 20 ml or more of PTSO was administered. The maximum response was obtained after 20 ml or more of PTSO was administered. It was also shown that milk yield was fully restored after 3-4 days, and the rate of recovery was dependent on the dose of PTSO. This indicated that the reduction in milk yield caused by PTSO was reversible and did not cause any damage to the udder (in ruminants).

[0246] These treatments open the possibility of preventing or reducing milk leakage and thus preventing new mastitis infections, and secondly, of reducing or preventing the painful udder pressure associated with the stress during the quick dry off. Therefore, administering PTSO to cows during the quick dry off is a valuable tool for cow health and dairy profitability.

[0247] Example 6. Synthesis of di-n-propyl thiosulfinate

[0248] Scope: This method describes how di-n-propyl thiosulfinate was synthesized. This compound was used to treat cows and its effect on milk yield was investigated.

[0249] Study Design: Under nitrogen atmosphere, in a three-neck flask, di-n-propyl disulfide (139.5 g, 1 Eq, 928 mmol) was dissolved in 460 ml of dichloromethane (DCM) and cooled to approximately 0-2° C. (internal). Metachloroperoxybenzoic acid (m-CPBA) (228.8 g, 70 wt %, 1.00 Eq, 1.33 mol) was dissolved in DCM (2.3 L) and the solution was added dropwise to the chilled di-n-propyl disulfide solution over 6 hours, keeping the temperature below 5° C. After complete addition, the reaction mixture was filtered and the solvent was evaporated under reduced pressure at approximately 30° C. to give a colorless oil. The oil was dissolved in isopropyl acetate (IPAc) (1 L) and the mixture was washed with aqueous NaHCO3 (5%, 6x, 500 mL), water (2x 300 mL) and brine (1x, 400 mL), dried over Na2SO4, filtered and evaporated at approximately 30°C to give a yellowish oil (144 g), which was purified by automated column chromatography (1.6 kg silica, 0-7% IPAc:heptane, runtime 5.5 h, 250 mL / min) to give PTS as a yellowish oil (122.4 g). This was again dissolved in IPAc (1 L), washed with aqueous NaHCO3 (5%, 6x, 500 mL), water (2x 300 mL) and brine (1x, 400 mL), dried over Na2SO4, filtered and evaporated at approximately 30 °C to give PTS (100 g, 65%) as a yellowish oil, which is related to the structure 1 It was analyzed by 1 H-NMR and its purity was analyzed by LCMS. 1H-NMR (400MHz, CDCl3): δ 3.18~3.00(m,4H),1.90~1.72(m,4H),1.06(t,3H,J=7.2Hz),1.01(t,3H,J=7.6Hz)ppm LCMS: Column: Zorbax SB-C8 (2.1×50 mm; 1.8 μm; RRHD 1200 bar). Flow rate: 0.6 ml / min, wavelength (λ): 215 and 246 nm. Mobile phase A: 10 mM NH4OAc (water / methanol / acetonitrile = 900 / 60 / 40) Mobile phase B: 10 mM NHOAc (water / methanol / acetonitrile = 100 / 540 / 360) Method: Mobile phase A / Mobile phase B: 95 / 5 (0.0 min ~ 2.0 min), 0 / 100 (1.5 min)

[0250] Results and Conclusions: 100 g of PTS was synthesized with a yield of 65% and a purity of 97.6% (a / a), λ 215 nm and 98.7% (a / a), λ 246 nm.

[0251] Example 7. Dose-Response PTS Treatment and Milk Yield

[0252] In this example, it is shown that di-n-propylthiosulfinate (PTS), a compound chemically related to DPD, exhibited the same or similar effect on milk yield reduction as DPD. Furthermore, the correlation between milk yield reduction and various doses of PTS was determined.

[0253] Study design. Twelve cows (Holstein-Friesian) from one farm were used. The start of the dry period was 40-60 days before calving for all cows. The cows were milked twice a day at a fixed time, and cows with milk production of more than 12.5 liters per day 24 hours before PTSO administration were selected. PTS was synthesized as described in Example 6. No special measures were taken to promote dry period, such as changes in feed allocation or milking frequency. The cows were milked twice a day. The cows were housed in free stables. Pork gelatin capsules with PTS or placebo gel capsules with water were administered 7-14 days before the start of the dry period.

[0254] For this study, various amounts of synthetic PTS (>98% purity) were administered to cows with a milk yield of at least 12.5 kg per day. The following doses were applied: 0, 10, 20, 40 ml of pure PTS. For each dose, two cows were used.

[0255] Milk production was monitored for 14 days before the various doses were administered. The gel capsules were administered orally by bolus shooter, while the gel capsules for the placebo group were filled with tap water. The capsules were administered between the morning and afternoon milkings. On t=14, PTS-containing and placebo capsules were administered, and milk production was monitored over 48 hours.

[0256] Results: In Table 16 below, milk yield and percent change in milk yield after administration of various doses of PTS are presented. A positive value of "% decrease" indicates a decrease in milk yield, while a negative value indicates an increase in milk yield. The reference treatment, during which a capsule of water was administered instead of PTS, showed no effect after 24 and 48 hours. However, administration of 20 ml of PTS clearly decreased milk yield compared to the milk yield before treatment, and the decreasing effect was stronger when more PTS was administered.

[0257] [Table 15]

[0258] [Table 16]

[0259] Discussion and conclusions: This study showed that there was a dose-response curve between the dose of PTS and the reduction in daily milk yield. The effect was evident after 40 and 60 ml of PTS was administered, but it cannot be excluded that lower doses may result in reduced milk yield. Perhaps further reduction in milk yield could be achieved by administering higher doses.

[0260] This study shows that other organosulfur analogues from DPD can also be used to achieve a temporary reduction in milk yield, giving the cow more time to reduce its natural milk yield by other means, for example by feed adaptation. This study also shows that PTS does not damage the udder (in ruminants) to reduce milk yield, since the reduction in milk yield is temporary and recovers 3-4 days after administration. These treatments open the possibility of preventing or reducing milk leakage and thus preventing new mastitis infections, and secondly, of reducing or preventing the painful udder pressure associated with the stress of the sudden dry-off. Therefore, administering PTS during the sudden dry-off is a valuable tool for cow health and dairy profitability.

[0261] Example 8. Treatment with DPD Analogs and Milk Yield

[0262] Range. In this example, it is demonstrated that treatment with chemically related compounds of DPD shows the same or similar effect on milk production reduction as DPD. In this example, the analogs were used as shown in Table 17 below.

[0263] Study design: Thirty-six cows (Holstein-Friesian) from two farms were used. Their diet consisted of a total mixed ration (TMR) containing corn and grass silage, supplemented with soybeans and minerals.

[0264] The start of the dry period was 40-60 days before calving for all cows. Cows were milked with a milk robot. Cows producing more than 12.5 liters of milk per day 24 hours before PTSO administration were selected. No special measures were taken to promote dryness, such as changes in feed allocation or milking frequency. The cows were milked twice daily. Cows were housed in free stalls. Pork gelatin capsules with the title compound or placebo gel capsules with water were administered 7-14 days before the start of the dry period. Placebo treatment corresponded to the administration of two gel capsules containing 180 ml of water.

[0265] For this study, the synthetic compounds (>96% purity) were administered to cattle at the doses shown in Table 17 below. The purpose of this study was to compare the activity of the corresponding doses of compounds expressed in moles based on the number of moles present in 80 ml of di-n-propyl disulfide at 20° C. The number of moles was converted to milliliters (ml) or grams of test compound. For this purpose, molecular weight and specific density were used in the calculations. The dosages of the pure organosulfur analogs are shown in Table 17 below. The compounds are formulated into gel capsules as shown in the previous examples.

[0266] Milk production was monitored for at least 5 days before the various doses were administered. The gel capsules were administered orally by bolus shooter, and the gel capsules for the placebo group were filled with tap water. The capsules were administered between the morning and afternoon milkings. On day t=14, the capsules and placebo capsules were administered, and milk production was monitored for over 96 hours.

[0267] Results. In the following table 17, milk yield and the relative change (%) in milk yield are presented. The positive value of "decrease %" indicates the decrease in milk yield, while the negative value indicates the increase in milk yield. In the reference treatment, during which capsules of water are administered instead of the compound, the decrease in milk yield is observed for all the compounds tested, although to a greater or lesser extent. Disulfides showed a significant effect, as did dialkyl (mono)sulfides, dialkyl trisulfides, dialkyl thiosulfinates, dialkyl thiosulfonates, and dialkyl sulfones.

[0268] The reduction in milk yield was temporary, with almost complete recovery after 96 hours, demonstrating that the reduction in milk yield due to administration of the compound was reversible and did not cause irreversible harm to the udder (in ruminants).

[0269] A further observation from these experiments concerns odor during treatment. A relationship was observed between the degree of odor generated and the number of sulfur atoms in the therapeutic compound. For example, di-n-propyl trisulfide resulted in a stronger odor than dipropyl disulfide, which in turn resulted in a stronger odor than dipropyl monosulfide. Therefore, compounds with a single sulfur group are preferred.

[0270] These treatments open the possibility of preventing or reducing milk leakage and thus preventing new mastitis infections, and secondly, of reducing or preventing the painful udder pressure associated with the stress during the quick dry-off. Therefore, administering these compounds to cows during the quick dry-off is a valuable tool for the health of the cows and the profitability of the dairy farm.

[0271] [Table 17-1] [Table 17-2] [Table 17-3]

[0272] [Table 18]

[0273] Example 9. Synthesis of dibenzyl thiosulfinate, dibenzyl thiosulfonate, and diisopropyl thiosulfonate

[0274] 9.1 Synthesis of dibenzyl thiosulfinate

[0275] Scope: This method describes how n-dibenzylthiosulfinate was synthesized. This compound was used to treat cows and its effect on milk yield was investigated.

[0276] Study Design: The synthesis of dibenzylthiosulfinate (CAS 16302-98-0) was described in Bioorganic & Medicinal Chemistry Letters., 2010, 5541-5543.

[0277] In a 2 L three-neck flask, 1,2-dibenzyldisulfane (40.0 g, 1 Eq, 162 mmol) and dichloromethane (2.1 kg, 1.6 L, 1.5 e + 2 Eq, 25 mol) (T int = 19 °C); 3-chlorobenzoperoxyacid (40.0 g, 77 wt %, 1.1 Eq, 179 mmol) was added in portions to the mixture, allowing the internal temperature (T int) was kept at room temperature. The solution was stirred at room temperature. After a few minutes, the colorless solution turned purple. After stirring at room temperature for 1.0 h, the reaction was quenched by adding approximately 200 mL of saturated sodium bicarbonate solution. The mixture was stirred for 5 min and the layers were separated. The organic layer was dried over sodium sulfate, filtered on a short pad of silica gel (glass filter P4), and concentrated under vacuum at 30 °C. Before complete evaporation of the solvent, 17 g of the previous batch was added (92% purity). After complete removal of the solvent, the pink solid (31 g) was stirred with 200 mL of EtOH at room temperature overnight, followed by filtration on a P3 glass filter. The solid was rinsed with 200 mL of pentane, the filter cake was dried on the filter, and subsequently dried under vacuum at 25 °C for 30 min to give dibenzylthiosulfinate as a dry solid. The product was kept as a dry solid and analyzed directly by HPLC after sample preparation. HPLC (column: Waters Xselect CSH C18, 2.1 × 50 mm, 2.5 μm; gradient: 10 mM (NH4)HCO3 / acetonitrile: 95 / 5 (0.5 min-4.0 min), 2 / 98 (0.5 min)): purity 97.5%, R t 3.189 minutes.

[0278] The cows were administered a freshly prepared formulation containing dibenzylthiosulfinate.

[0279] 9.2 Synthesis of dibenzylthiosulfonate

[0280] Scope. This method describes how dibenzylthiosulfonate was synthesized. This compound was used to treat cows and its effect on milk yield was examined.

[0281] Experimental design. A 1 L round-bottomed three-neck flask (equipped with an air condenser, magnetic stirring egg, temperature sensor and dropping funnel) was charged with 1,2-dibenzyl disulfide (70 g, 284 mmol, 1 equiv.) and acetic acid (244 mL). The suspension was magnetically stirred (internal temperature: 21° C.). Hydrogen peroxide (35% solution in water, 63.5 mL, 739 mmol, 2.6 equiv.) was added dropwise to the suspension over 5 min. The mixture was stirred at ambient temperature overnight for 16 h without cooling, and a thick slightly orange suspension was obtained at a temperature of 21° C. IPC-LCMS-22 showed complete conversion of the starting material. Water (240 mL) was added to the mixture, and after 10 min the suspension was filtered. The solid was washed with water (2 x 50 mL), then cold EtOH (50 mL) and dried in vacuo to give a slightly orange solid (59 g, 75% yield). LCMS-5 purity 79% (@222 nm).

[0282] The crude material was combined with 10 g from a previous experiment and the combined amount was dissolved in boiling EtOH (350 mL, 5 vol). The solution was allowed to cool slowly overnight with stirring. Crystals formed after approximately 5 min. The mixture was further cooled in ice water for 30 min and then filtered. The solid was washed with cold EtOH (40 mL) and dried in vacuo to give 55.7 g of an off-white crystalline solid with LCMS-5 purity of 92%.

[0283] The solid was recrystallized a second time from boiling ethanol (450 mL) and iPrOAc (20 mL). The hot solution was seeded and aged at 50° C. for 2 h, and then stirred at ambient temperature overnight. The suspension was filtered and the solid was washed with cold EtOH and dried in vacuo to give dibenzylthiosulfonate as a white solid (48.2 g, 70% crystallization recovery). NMR: 1H-NMR (400 MHz, CDCl3) δ 7.44-7.22 (m, 12H), 4.19 (s, 1H), 4.01 (s, 1H) (Conform structure). LCMS-5: tR 1.45 min. 98.6% purity at 222 nm. M / z+ 296.0 [M+18]. DSC: onset 107.45 °C, peak 108.53 °C. 98.9 mol% purity.

[0284] The cows were administered a freshly prepared formulation containing dibenzylthiosulfonate.

[0285] 9.3 Synthesis of diisopropyl thiosulfonate

[0286] Scope: This method describes how diisopropyl thiosulfonate is synthesized. This compound was used to treat cows and its effect on milk yield was investigated.

[0287] Experimental design

[0288] To a solution of 1,2-diisopropyldisulfane (95.0 g, 1 Eq, 632 mmol) in acetic acid (750 mL) was added hydrogen peroxide (129 g, 114 mL, 35 wt%, 2.10 Eq, 1.33 mol) dropwise. An ambient temperature water bath was used for external cooling. The temperature was monitored and kept below 44° C. by adding ice to the water bath. The reaction was 1 H-NMR was used to monitor.

[0289] After stirring overnight at room temperature, hydrogen peroxide (30.7 g, 27.1 mL, 35 wt%, 0.50 Eq, 316 mmol) was added dropwise. Stirring was continued for 16 h at 35° C. Then, another portion of hydrogen peroxide (61.4 g, 55.3 mL, 35 wt%, 1.00 Eq, 632 mmol) was added dropwise over 1 h and stirring was continued at approximately 36° C. for an additional 6 h.

[0290] Acetic acid was then distilled off under reduced pressure (very slowly) at 45°C to give a colourless oil (190.9 g). The crude oil was purified by column chromatography (800 g SiO2, using a gradient of heptane and ethyl acetate as eluent). Fractions were analysed by HPLC (column: Waters Xselect CSH): Waters Xselect CSH C18; gradient: 10 mM (NH4)HCO3 / acetonitrile: 95 / 5 (0.5 min to 4.0 min), 2 / 98 (0.5 min): and the main fractions were combined to give diisopropylthiosulfonate as a colourless oil (37 g, 33% yield) with a purity of 96.4% (a / a) (LC-UV, lampda 215 nm).

[0291] The cows were administered a freshly prepared formulation containing diisopropyl thiosulfonate.

[0292] Example 10. Administration of DPD and incidence of mastitis

[0293] Scope: Cows were orally administered 80ml DPD and 80ml onion extract containing DPD and the cows were monitored accordingly for the occurrence of mastitis.

[0294] Study design. Description of cows. Fifty-nine cows from five farms were used in the study. The cows were randomly assigned to three groups: - control group; - DPD treatment group 1: DC liquid (500 mL) group (80 ml of a composition containing pure dipropyl disulfide); and - DPD-treated Group 2: DPD-enriched onion Extract (OE) bolus (150 mL) group (equivalent to 80 ml of pure dipropyl disulfide).

[0295] Farm 1 (Belgium) has a total of 190 purebred Holstein Friesian (HF) cows, of which 165 are lactating and 25 are dry. The farm is milked three times a day in a 2*13 side by side milking parlor with display. Sensub allflex transponders are used for animal identification. Average milk yield is 37 liters per day with a 305 day production of 11800 kg. Somatic Cell Count (SCC) records are taken every 5 weeks. The cows are dry-dried off suddenly and have an average production of 26.5 kg. The cows are dry-dried off once a week after midday milking. The criterion for selective desiccation using antimicrobials is an SCC of more than 100,000. All cows are given an internal and external sealant. Dry cow housing consists of pens with rubber matrasses. The dry period is targeted to be 5 weeks for multiparous cows and 6 weeks for uniparous cows.

[0296] Farm 2 has 300 HF cows and 25 dry cows with an average SCC of 280,000 (Belgium). Cows are milked twice a day in a 2*20 side-by-side milking system with displays. Cows have individual identification by necklace. The farm has an average production of 32 kg and a 305-day production of 10,000 kg. Cows are dry-off suddenly and the farm uses a blanket antimicrobial treatment at dry-off. Individual SCC is performed four times a year and dry cows are housed in pens with matrasses.

[0297] Farm 3 has 146 HF cows in Belgium with 138 milking and 8 dry cows with an average SCC of 113,000 and an average production of 33.3 kg, fat of 4.65 and protein of 3.85, and a 305-day production of 10143 kg. Milking is done twice daily in a Midiline ML3100, 2*20 swing-over milking parlor, with SCC every 5 weeks. Cows are dry-off quickly and the farm uses a blanket antibacterial treatment at dry-off. Individual SCC is done 4 times a year and the dry cows are housed in pens with matrasses.

[0298] Farm 4 is a robotic milking farm with two robots located in the Netherlands. There are a total of 119 HF cows and 5 dry cows with an average milk production of 28.9 kg, fat content of 4.73 and protein content of 3.82.

[0299] Farm 5 is a robotic milking farm with 4 Lely robots and 245 HF cows (224 dairy and 21 dry cows in France). Average daily milk production is 29 litres.

[0300] The control group consisted of 24 cows with an average milk yield of 17.23 L before drying off. Eight cows had a milk production (mp) of 12.5 L or less before drying off (minimum: 6.50 L and maximum: 29.20 L). The average lactation days in the control group was 2.1 days, and they had an average dry period of 48 days (10 cows in the first lactation, 4 cows in the second lactation, 6 cows in the third lactation, and 4 cows in the fourth lactation). Milk leakage was observed in 50% of all cows in the control group, regardless of milk production before drying off, and the incidence of cows with a dry-off production of 12.5 L or less or of cows that were discarded increased to 63%.

[0301] Two DPD treatment groups

[0302] The DC liquid treatment group consisted of 18 cows with a mean mp of 21.61 L before drying off (one cow had an mp of less than 12.5 L, a low of 11.40 L, and a high of 32 L). The mean days in lactation in the DC liquid group was 1.9 days with 11 cows in their first lactation, 2 in their second lactation, 3 in their third lactation, and 2 in their fifth lactation. The mean dry off period for this group was 49 days.

[0303] The DPD-enriched OE bolus treatment group consisted of 17 cows with a mean mp of 20.6 L (min 13.8 L and max 32 L) before drying off and a mean length of lactation of 2.2 days (6 cows in 1st lactation, 4 cows in 2nd lactation and 4 cows in 3rd lactation). One cow dropped out during the dry off period due to non-related lameness issues. The dry off period for this group averaged 46 days.

[0304] In DPD treatment groups 1 and 2 (DC fluid and DPD-enriched OE), the combined incidence of milk leakage was 17%, which corresponds to a 73% reduction.

[0305] Cows are dried off about 6-7 weeks before expected calving. Most cows are dried off suddenly, which causes high udder pressure, open teat canals, and often milk leakage. As a result, mastitis-causing microorganisms can invade the teat canals and colonize the udder. After calving and lactation resumes, these microorganisms can become pathogenic and cause mastitis. Thus, cows are very vulnerable during this period. By administering DPD to cows just before the sudden dry off, it is hoped that the udder pressure will decrease and the teat canals will close, resulting in less or no milk leakage, and less mastitis-causing microorganisms will invade the udder.

[0306] After the last milking, the cows underwent a dry-off protocol according to the farm's protocol. The DPD treatment group received DPD treatment in the form of a bolus administered intraruminally via a bolus shooter. Both treatment groups consisted of treatment with 80 ml of DPD. In all cases, a sudden dry-off was applied, with no restrictions on the number of milkings or on the replacement of feed before drying off. All cows had their hooves trimmed before being transferred to the dry pen. The cows were monitored for signs of mastitis during the dry period and after calving. Before the cows were in lactation for 30 days, SCC was determined to assess the dry period.

[0307] Intramammary infection (IMI) was defined as cases of clinical signs of mastitis (e.g. flocks, watery milk, coloring of milk) during the dry period and during the first 30 days of lactation, or cases of an increase in individual somatic cell count (ISCC) from before to after the dry period (threshold: 200,000 cells / mL), or both.

[0308] Results and Discussion

[0309] During the dry period, none of the cows showed any characteristics of mastitis. In Figure 9, the number of cows with mastitis symptoms and the incidence of cows diagnosed with mastitis are summarized. During the dry period, one cow in the DPD treatment group showed signs of clinical mastitis. This cow entered the dry period with an increased cell count. After calving, signs of mastitis (flocs in milk) were observed in one cow in the DPD treatment group. No clinical signs of illness, such as fever and / or lethargy, were observed and the cow recovered naturally. Bacterial sampling detected low pathogenicity E. coli.

[0310] In the control group, after calving, 73% of cows had elevated SCC, while 73% of the animals were dried off with antibiotics. One cow was diagnosed with intramammary infection (SCC >200,000 before drying off) and showed a decrease in cell count to <200,000 at the start of lactation. 36% of the animals were diagnosed with new IMI.

[0311] In the DPD-treated group, a postpartum reduction in SCC was identified in 61% of cases. In particular, in the DC liquid group, a postpartum reduction in SCC was identified in 55%. In the DPD-rich OE group, a decrease in SCC was identified in 67% of animals. Three animals (two in the DC liquid group and one in the DPD-rich OE group) were diagnosed with IMI and dried off, but all three had recovered at the start of the new lactation. After parturition, 21% of animals were newly diagnosed with IMI (27% in the DC liquid-treated group and 16% in the DPD-rich OE-treated group).

[0312] In the DPD-treated group, the total incidence of new IMI was 21%, corresponding to a 42% reduction in the risk of new IMI.

[0313] As demonstrated in Figure 9, cows treated with DPD at the dry off period have a reduced risk of new intramammary infections. This benefits the health and wellbeing of the cows as well as the dairy farmer. The cows are at reduced risk for subclinical and clinical infections, while the dairy farmer is at reduced risk of exceeding the capacity of the high bulk milk cell tank with the corresponding reduced expenditure on veterinary care. Cows that do not recover well during the dry off period become chronic and are often removed from the dairy farm.

[0314] The study concluded that the incidence of new intramammary infections was reduced when cows were treated with DPD immediately prior to the sudden dry-off.

[0315] Another advantage is that the composition and amount of the ration is adjusted in the last week before the sudden dry-off occurs. These ration adjustments reduce milk yield to some extent, but do not impair the health of the animals. This is done deliberately in accordance with the Mastitis Council guidelines, which recommend drying off cows with a milk production of 12.5 kg or less (mp) to reduce the risk of milk leakage. Until the ration is reduced to the sudden dry-off, these ration adaptations result in a reduction in milk yield equivalent to 50 euros per high-producing cow per day. If DPD or a composition containing sufficient DPD is used before the sudden dry-off, these ration adjustments are not necessary, resulting in benefits for the dairy farmer.

Claims

1. a) in reducing lactation in mammals; b) In the prophylactic treatment of intramammary infections in mammals and / or in reducing the incidence of dry-off-related stress, dry-off-related inflammation or dry-off-related infection; and / or, c) In promoting the health and well-being of lactating mammals A preparation containing di-n-propylthiosulfonate (PTSO) or di-n-propylthiosulfinate (PTS) for use, PTS is administered in a dose of at least 18 g, or PTS is administered in a dose of at least 20 g. The aforementioned agent.

2. a) in reducing lactation in mammals; b) In the prophylactic treatment of intramammary infections in mammals and / or in reducing the incidence of dry-off-related stress, dry-off-related inflammation or dry-off-related infection; and / or, c) In promoting the health and well-being of lactating mammals For use, an agent containing a compound conforming to the following formula I: 【Chemistry 1】 Equation I Here, R 1 and R 2 C 1～4 Selected from the group consisting of alkyl and benzyl; where preferably, the C 1～4 Alkyl is methyl, ethyl, n-propyl, isopropyl, or n-butyl; where C 1～4 Alkyl and benzyl may be unsubstituted.

3. R 1 and R 2 The agent according to claim 2, wherein the two are identical.

4. The agent according to claim 2, wherein the compound is selected from di-n-propyl disulfide, dimethyl disulfide, diethyl disulfide, diisopropyl disulfide, di-n-butyl disulfide, and dibenzyl disulfide.

5. The agent according to claim 2, wherein the compound is di-n-propyl disulfide.

6. A method for reducing lactation in a non-human mammal; for the prophylactic treatment of intramammary infections in a non-human mammal and / or for reducing the incidence of dry-off-related stress, dry-off-related inflammation or dry-off-related infection; or for promoting the health and well-being of a lactating non-human mammal, comprising administering at least 18 g of di-n-propylthiosulfonate (PTSO) or at least 20 g of di-n-propylthiosulfinate (PTS) to a lactating non-human mammal.

7. A method for reducing lactation in a non-human mammal; for the prophylactic treatment of intramammary infections in a non-human mammal and / or for reducing the occurrence of dry-off-related stress, dry-off-related inflammation or dry-off-related infection; or for promoting the health and well-being of a lactating non-human mammal, the method comprising administering to a lactating non-human mammal a pharmaceutical or animal composition or functional food composition comprising a compound according to the following formula I: 【Chemistry 2】 Equation I Here, R 1 and R 2 are each independently selected from the group consisting of C 1～4 alkyl and benzyl; wherein preferably, said C 1～4 alkyl is methyl, ethyl, n-propyl, isopropyl or n-butyl; wherein said C 1～4 alkyl and benzyl may be unsubstituted.

8. The method according to claim 7, wherein the non-human mammal is a ruminant, preferably a cattle.

9. The method according to claim 7, wherein R1 and R2 are the same.

10. The method according to claim 7, wherein the compound is selected from di-n-propyl disulfide, dimethyl disulfide, diethyl disulfide, diisopropyl disulfide, di-n-butyl disulfide, and dibenzyl disulfide.

11. The method according to claim 7, wherein the compound is di-n-propyl disulfide.

12. The method according to claim 7, wherein the compound is formulated as a single-dose unit comprising at least 50 grams, preferably at least 70 grams, of the compound, and the composition is administered to a pregnant ruminant.

13. The method according to claim 7, comprising selecting a pregnant cow that produces at least 10 liters of milk per day, and administering the composition to the cow.

14. The method according to claim 7, wherein milking is abruptly or gradually stopped in the non-human mammal, and the compound is administered before or on the day the milking is stopped.

15. A pharmaceutical or veterinary composition or functional food composition containing a compound according to the following formula I: 【Transformation 3】 Equation I Here, R 1 and R 2 C 1～4 Selected from the group consisting of alkyl and benzyl; where preferably, the C 1～4 Alkyl is methyl, ethyl, n-propyl, isopropyl, or n-butyl; where C 1～4 Alkyl and benzyl may be unsubstituted.

16. The composition according to claim 15, wherein the compound is formulated as a single-dose unit comprising at least 50 grams of the compound, preferably at least 70 grams of the compound.

17. R 1 and R 2 The composition according to claim 15, wherein the two elements are identical.

18. The composition according to claim 15, wherein the compound is selected from di-n-propyl disulfide, dimethyl disulfide, diethyl disulfide, diisopropyl disulfide, di-n-butyl disulfide, dibenzyl disulfide, diethyl sulfide, di-n-propyl sulfide, diisopropyl sulfide, di-n-butyl sulfide, diphenyl sulfide, dibenzyl sulfide, di-n-propyl trisulfide, di-n-propyl sulfone, dibenzyl thiosulfinate, dibenzyl thiosulfonate, diisopropyl thiosulfonate, di-n-propyl thiosulfonate (PTSO), and di-n-propyl thiosulfinate (PTS).

19. The composition according to claim 15, wherein the compound is di-n-propyl disulfide.

20. The composition according to claim 15, further comprising a dry-off agent, preferably a prolactin inhibitor, such as cabergoline and quinagolide; casein hydrolysate; or a dry-off agent selected from acid-producing mineral boluses.

21. The composition according to claim 15, wherein the composition is a gel capsule.

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