HK1023288A - An oil composition of dihydropolyprenols - Google Patents

Description

Oil composition of dihydropolyprenol

Technical Field

The present invention relates to a novel composition for parenteral administration, and more particularly, to an oil composition of dihydropolyprenol for enhancing the immune system of animals.

Background

Dihydropolyprenols are a known class of polyprenols. It has now been found that such compounds enhance the resistance of non-specific hosts to a variety of pathogenic bacteria. Such hosts include rodents, cows, calves, piglets and pigs. The compounds are useful as agents for treating or controlling animal and human infections by increasing host resistance.

It is becoming increasingly apparent, however, that the development of pharmaceuticals is far beyond the scope of finding only highly biologically active compounds, and that many other aspects must be considered before highly potent active compounds can be formulated into pharmaceutical products, the most important of which are the stability of the active agent, the ability to be absorbed from the site of administration, the physical state of the active agent, and many other related problems, economic factors such as the cost of large quantities of the pharmaceutical, in addition to the properties of the active agent itself.

In view of these problems, the present invention provides an unexpected dihydropolyprenol oil composition which is pharmaceutically elegant and avoids all of the above-mentioned problems. Such oil compositions limit the components to only: excipients (i.e., oils) and, effectively, dihydropolyprenols. Since there are few components which can adversely react with the dihydropolyprenol, it can be stored for a long period of time. It is known that "oxidation" is the main route of degradation of the polyprenols, whereas oil compositions protect the dihydropolyprenols from oxidation, since oxygen must pass through the oil vehicle by diffusion to reach the dihydropolyprenols, and it is more advantageous that some oils contain endogenous antioxidants. In addition, the physical stability of the oil composition is not problematic because it is a single-phase solution. On the other hand, other related formulations such as emulsions disclosed in the related art are biphasic, possibly reacting with each other over time.

The oil composition of the present invention is easy to administer because it can be formulated more concentrated than an emulsion, thereby allowing a smaller injection volume to be administered to an animal. For example, emulsions are typically prepared at a concentration of 10mg/ml, whereas oil-based formulations of the invention may have a concentration of at least 150mg/ml, the volume of which is only 1/15 of the emulsion. The oil composition is also easy to inject because its viscosity is low enough to flow through a conventional syringe and needle, whereas, for example, an implantable pellet containing dihydropolyprenol is difficult to administer to an animal.

Finally, a simple process for the large-scale production of dihydropolyprenol oil compositions is obtained, while the processes for preparing other dosage forms tend to be more complicated and costly.

Information disclosure

US4,624,966 discloses β, γ -dihydropolyprenol derivatives useful as immunizing agents against infectious diseases in humans and animals and methods of use thereof.

US4,839,389 discloses an injection containing a polyprenol, the main components of which are polyprenol and lecithin.

US5,139,740 discloses a soft capsule composition comprising a polyisoprene alcohol compound, a surfactant and/or an unsaturated aliphatic acid.

US5,280,048 discloses a method of combating infection with polyisoprene alcohol compounds.

Other reference materials of general interest are:

nagahata, g.j.kociba, h.noda, m.koiwa, and m.kimura, "effect of dihydroheptaprenyl alcohol on neutrophil function in post-partum cows", "Veterinary Immunology and Immunopathology" (Veterinary Immunology and Immunopathology), 29, pp.163-169 (1991).

O.Yoneyama, S.Osame, S.Ichijo, M.Kimura, S.Araki, M.Suzukian E.Imamura, "Effect of dihydroheptaprenyl alcohols on neutrophil function in calves", British Veterinary journal (British Veterinary Kouranl, 145, pp.531-537 (1989)).

O.Yoneyama, S.Osame, M.Kimura, S.Araki and S.Ichijo, "enhancement of neutrophil function in adult cows with dihydroheptaprenyl alcohol," -Journal of Veterinary Science, 51(6), PP.1283-1286 (1989).

T.Watari, R.Goitsuka, H.Koyama, T.Sako, T.Uchino, S.Araki, A.Hasegawa and S.Motoyoshi, "Effect of dihydroheptaprenol on reduction of nitroblue tetrazolium (NBT) by porcine dust cells", "Journal of veterinary medicine in Japan, 51(3), pp.630-631 (1989)".

S. Araki, M.Suzuki, K.Ogura, M.Kimura, E.Imamura, C.Kuniyasu, K.Kagaya and Y.Fukazawa, "enhancing the phagocytic and bactericidal activity of neutrophils in piglets with dihydroheptaprenyl alcohols, a synthetic polyprenol derivative", "Microbiology and immunology (Microbiology, Immunol.),33(10), pp.877-882 (1989).

S. Araki, K.Kagaya, K.Kitoh, M.Kimura and Y.Fukazawa, "enhancing mice against E.coli Infection with dihydroheptaprenyl alcohols, a synthetic polyprenol derivative", Infection and Immunity (Infection and Immunity), 55(9), pp.2164-2170 (1987).

M. Kimura, S, Araki, T.Nakai and K.Kume, "protection of experiments with Dihydropolyprenyl alcohols in combination with vaccines on guinea pigs and piglets infected with A.pneumoniae,", (J.J.Japanese of Veterinary Science, 55 (4)), pp.627-630 (1993).

E.J.Robb, D.D.Kratzer, C.H.Ho and K.J.Dame, "dosage characterization and dosage form evaluation of dihydroheptaprenyl alcohols during induced bovine pasteurellosis pneumoniae" [ Journal of Animal Science 71 (Suppo 1.1), p.203(1993) ].

Summary of the invention

It is an object of the present invention to provide a pharmaceutical composition comprising a dihydropolyisoprene alcohol and an oleaginous excipient which enhances the intrinsic anti-infective properties of animals and stimulates the immune system, and which is biocompatible with respect to animals.

It is another object of the present invention to provide an oil composition containing a dihydropolyprenol at a high concentration so that its one injection volume is convenient for administration.

It is still another object of the present invention to provide an oil composition having physical and chemical stability which can maintain a suitable shelf life when the composition is contained in an economical "ready-to-use" container.

It is a further object of the present invention to provide a low viscosity oil composition so that the composition is easy to inject.

It is a further object of the present invention to provide an oil composition which is simple to manufacture so as to minimize the final cost of the product.

The object of the present invention has been achieved by the following technical solutions. The present invention provides an oil composition for parenteral administration which comprises essentially a dihydropolyisoprene alcohol of formula I wherein n is an integer from 5 to 7 and a pharmaceutically acceptable oil.

Detailed Description

Surprisingly and unexpectedly, it has been found that such substantially water-free oil compositions of the present invention improve the utilization of the dihydropolyprenols of formula I. Such compositions provide a stable, easy to administer and simple to manufacture delivery vehicle.

Dihydropolyprenols are a known class of compounds. Their activity in enhancing the immune system of animals and methods of preparation are disclosed in US4,624,966; US4,839,389; US5,139,740; US5,280,048; which is incorporated herein by reference.

The preferred dihydropolyprenol of formula i for preparing the oil composition of the present invention is dihydroheptaprenol, a compound of formula i wherein n = 6.

Animals refer to livestock, poultry and companion animals, but the invention also applies to other vertebrates, as well as other lower species including invertebrates.

The oil composition of the present invention can be prepared by mixing a dihydropolyprenol of formula I with a pharmaceutically acceptable oil. Methods for preparing such compositions are conventional and well known to those skilled in the art. For example, a desired weight or volume of oil is placed in a glass or stainless steel container. The oil was filtered through a sterile filter (0.22 micron filter) into the container. The weight or volume of the dihydropolyprenol used for preparing the preparation of a certain concentration was calculated, and it was poured into the above oil container and stirred to obtain a uniform mixture. The time required for agitation depends on the size of the vessel used and the type of mixing device. Typically 100ml batches are stirred for 5 minutes to obtain a homogeneous solution. While a 1000 liter batch requires about 30 minutes of stirring. The resulting homogeneous solution was filtered through a sterile filter (0.22 micron filter) and filled into vials.

The term "pharmaceutically acceptable" refers to those properties which are biocompatible with the subject from a pharmacological/toxicological point of view and which are stable, soluble and bioavailable from a physical/chemical point of view by the pharmacist making the drug.

The pharmaceutically acceptable oil is coconut oil.

"Miglyol 810", "Miglyol 812" and "Miglyol 829" are trademarks of Huls corporation as Miglyol , separate and refined grades of coconut oil, containing primarily medium chain triglycerides.

The route of administration of the oil composition of the present invention is parenteral administration. The preferred route of administration to livestock is subcutaneous administration so as not to damage the edible tissues. But may also be administered by intramuscular or other parenteral routes of administration.

The amount of dihydropolyprenol of formula I and the concentration of the oil composition used in the present invention can be varied and adjusted over a wide range depending on the potency of the particular compound used, the desired dosage, the desired concentration, and/or the particular animal being treated. Generally, the active ingredient is used in an amount of between 0.1mg/kg and 1000mg/kg and at a concentration of between 1mg/ml and 500 mg/ml. The upper concentration limit is reached when the oil composition is too viscous to be injected. For example, the dihydroheptaprenyl alcohol is used in a dose of 1 to 10mg/kg for the prevention of respiratory diseases (BRD) in cattle in breeding facilities. For example, a solution concentration of 150mg/ml is preferred when the BRD treatment is carried out on cattle in a farm with a 2mg/kg dose of dihydroheptaprenyl alcohol. Thus, 1.3ml of the dihydropolyprenol required for the treatment of ordinary animals can be provided by one injection.

The oil composition of the present invention will be better understood by reference to the following examples. These examples are intended to be illustrative and not limiting of the scope of the invention. It is believed that one skilled in the art can, without further elaboration, utilize the preceding description to provide further guidance in practicing the invention, including the information provided in the examples set forth herein.

Example 1

0.341g of dihydroheptaprenyl alcohol was added to a 20ml glass beaker containing 10.997g of Miglyol 812. The solution was stirred with an electromagnetic stir bar for 30 minutes. The solution was then added to a 5ml glass vial, stoppered, capped and labeled. The concentration of the obtained dihydroheptaprenol was 30.1mg/g of the preparation. Challenge dose with haemolytic pasteurella in a whole body protection assay in mice (28.2 half lethal dose, LD)₅₀) To test this formulation.

Example 2

0.339g of dihydroheptaprenyl alcohol was added to a 20ml glass beaker containing 10.996g of Miglyol 812. The solution was stirred with an electromagnetic stir bar for at least 30 minutes. The solution was then added to a 5ml glass vial, stoppered, capped and labeled. Process for producing dihydroheptaprenyl alcoholsThe concentration was 26.5mg/ml of the formulation. Challenge dose with pasteurella haemolytica (12 LD) in a whole body protection assay in mice₅₀) To test this formulation.

Example 3

1.886g of the product of example 2 was diluted with 26.467g of Miglyol810 and the two solutions were mixed in a beaker for 10 minutes. The concentration of the obtained dihydroheptaprenol was 9.8 mg/ml. Challenge dose (10 LD) with Pasteurella haemolytica in a systemic protection assay in mice₅₀) To test this formulation.

Example 4

0.1g of dihydroheptaprenyl alcohol was put into a 20ml glass vial, followed by 9.9g of Miglyol 810. The vial was vortexed using a Yamato vortexer for 1 minute at full speed. After 1 hour of standing, the vial was again vortexed for 15 seconds, stoppered, capped, and labeled. The concentration of the obtained dihydroheptaprenol was 10mg/g of the preparation. Challenge dose with pasteurella haemolytica (22 LD) in a whole body protection assay in mice₅₀) To test this formulation.

Example 5

0.1g of dihydroheptaprenyl alcohol was put into a 20ml glass vial, followed by 9.9g of Miglyol 812. The vial was vortexed using a Yamato vortexer for 1 minute at full speed. After 1 hour of standing, the vial was again vortexed for 15 seconds, stoppered, capped, and labeled. The concentration of the obtained dihydroheptaprenol was 10mg/g of the preparation. Challenge dose with pasteurella haemolytica (22 LD) in a whole body protection assay in mice₅₀) To test this formulation.

Example 6

0.1g of dihydroheptaprenyl alcohol was put into a 20ml glass vial, followed by 9.9g of Miglyol 829. The vial was vortexed using a Yamato vortexer for 1 minute at full speed. After 1 hour of standing, the vial was again vortexed for 15 seconds, stoppered, capped and addedAnd (4) a label. The concentration of the obtained dihydroheptaprenol was 10mg/g of the preparation. Challenge dose with pasteurella haemolytica (22 LD) in a whole body protection assay in mice₅₀) To test this formulation.

Example 7

0.1g of dihydroheptaprenyl alcohol was put into a 20ml glass vial, followed by addition of 9.9g of safflower oil. The vial was vortexed using a Yamato vortexer for 1 minute at full speed. After 1 hour of standing, the vial was again vortexed for 15 seconds, stoppered, capped, and labeled. The concentration of the obtained dihydroheptaprenol was 10mg/g of the preparation. Challenge dose with pasteurella haemolytica (22 LD) in a whole body protection assay in mice₅₀) To test this formulation.

Example 8

0.1g of dihydroheptaprenyl alcohol was put into a 20ml glass vial, followed by addition of 9.9g of sesame oil. The vial was vortexed using a Yamato vortexer for 1 minute at full speed. After 1 hour of standing, the vial was again vortexed for 15 seconds, stoppered, capped, and labeled. The concentration of the obtained dihydroheptaprenol was 10mg/g of the preparation. Challenge dose (25 LD) with Pasteurella haemolytica in a systemic protection assay in mice₅₀) To test this formulation.

Example 9

0.1g of dihydroheptaprenyl alcohol was added to a 20ml glass vial, followed by 9.9g of soybean oil. The vial was vortexed using a Yamato vortexer for 1 minute at full speed. After 1 hour of standing, the vial was again vortexed for 15 seconds, stoppered, capped, and labeled. The concentration of the obtained dihydroheptaprenol was 10mg/g of the preparation. Challenge dose with haemolytic pasteurella in a whole body protection assay in mice (6 LD)₅₀) To test this formulation.

Example 10

0.1g of dihydroheptaprenyl alcohol is addedA20 ml glass vial was then charged with 9.9g of cottonseed oil. The vial was vortexed using a Yamato vortexer for 1 minute at full speed. After 1 hour of standing, the vial was again vortexed for 15 seconds, stoppered, capped, and labeled. The concentration of the obtained dihydroheptaprenol was 10mg/g of the preparation. Challenge dose with pasteurella haemolytica (9 LD) in a whole body protection assay in mice₅₀) To test this formulation.

Example 11

0.1g of dihydroheptaprenyl alcohol is placed in a 20ml glass vial, followed by 9.9g of peanut oil. The vial was vortexed using a Yamato vortexer for 1 minute at full speed. After 1 hour of standing, the vial was again vortexed for 15 seconds, stoppered, capped, and labeled. The concentration of the obtained di-heptahydropolyprenol was 10mg/g of the formulation. Challenge dose (25 LD) with Pasteurella haemolytica in a systemic protection assay in mice₅₀) To test this formulation.

Example 12

0.1g of dihydroheptaprenyl alcohol is placed in a 20ml glass vial, followed by 9.9g of mineral oil. The vial was vortexed using a Yamato vortexer for 1 minute at full speed. After 1 hour of standing, the vial was again vortexed for 15 seconds, stoppered, capped, and labeled. The concentration of the obtained dihydroheptaprenol was 10mg/g of the preparation. Challenge dose with pasteurella haemolytica (9 LD) in a whole body protection assay in mice₅₀) To test this formulation.

Example 13

The formulations are prepared in a laminar flow hood using reliable aseptic techniques. 9.02g of dihydroheptaprenyl alcohol which had been filtered through a 0.22 μm filter and 47.73g of Miglyol810 were placed in a 100ml sterilized glass beaker and stirred for 10 minutes with an electromagnetic stir bar. The solution was then added to a 50ml sterile glass vial, stoppered, capped and labeled. The measured concentration was 142.2 mg/ml. Placebo was prepared by filtering approximately 50ml of miglyol810 through a 0.22 micron filter into 50ml sterile glass vials, stoppering, capping and labeling. This formulation was tested in the pasteurella haemolytica bovine immunity test.

Example 14

The formulations are prepared in a laminar flow hood using reliable aseptic techniques. 21.43g of dihydroheptaprenyl alcohol and 110.94g of Miglyol810 filtered through a 0.22 μm filter were placed in a 200ml sterile glass beaker and stirred for 10 minutes with an electromagnetic stir bar. The solution was then added to a 50ml sterile glass vial, stoppered, capped and labeled. The measured concentration was 149.8 mg/ml. Placebo was prepared by filtering approximately 50ml of miglyol810 through a 0.22 micron filter into 50ml sterile glass vials, stoppering, capping and labeling. This formulation was tested in the pasteurella haemolytica bovine immunity test.

Example 15

Whole body protection test for mice

Each of the compositions of examples 1-12 was evaluated in a Pasteurella haemolytica mouse systemic protection assay. In this test, each mouse was given a dose of the composition by subcutaneous injection. After 24 hours the challenge was performed with pasteurella haemolytica. The challenge dose in example 1 is the dose required to kill 50% of mice (LD)₅₀=28.2 times of 28.2). The challenge doses for the other examples are listed in table 1. The dosage varies from 25 to 400 mg/kg/day. Each composition was diluted with the measured amount of oil prior to dosing so that its one-shot volume was suitable for rats. A group receiving 0mg/kg was injected with Miglyol812 as a control. The haemolytic pasteurella species used was UC6531, originally isolated from a bovine respiratory case. The test strains were stored on 3mm glass beads immersed in 10% glycerol in casein tryptic hydrolysate soy medium (Becton-Dickinson Microbiol systems) and maintained at-70 ℃ until revitalisation. The number of mice survived after 7 days was recorded, and the median effective dose ED was calculated from the number of survived mice₅₀. Table 1 summarizes the test results.

Example 16

Bovine immunity test of pasteurella haemolytica

The composition of example 13 was evaluated in a pasteurella haemolytica bovine immunity test. In this test, 35-50 day old Holstein bull calves were randomly assigned to each treatment group by weight. 15 animals were assigned to either the placebo group receiving only the Miglyol810 excipient or the group receiving the 4mg/kg oil formulation. Each animal was given 4mg/kg subcutaneously and challenge experiments were performed 24 hours later. Challenge test included intratracheal inoculation of 6X 10⁶Colony Forming Units (CFU)/toxic haemolytic Pasteurella calves (A1) (dissolved in 10ml of acidified (pH4.5) Brain Heart Infusion (BHI) broth), challenge test was carried out 4 hours later for each calf with 4X 10⁸CFU of pasteurella haemolytica (dissolved in 10ml of non-acidified BHI broth). Observing calf for 10 days, weighing, and performing cadaver test. The judgment is based on the calculated scores of the mortality, the number of calves with the weight gain of 2.27kg during the research period and the lung consolidation. The experiment was designed to have the ability to detect 80% of the mortality and growth rate differences of α =0.2, 21-23%.

None of the calves died in the group receiving 4mg/kg and a clear difference in the placebo group, 2 of the 15 calves died (P =0.04, unilateral). It is evident that more calves (P =0.006, unilateral) gained 2.27kg (14 out of 15) in the 4mg/kg group compared to the negative control (7 out of 15). There was no significant difference in lung lesion scores.

The composition of example 14 was evaluated in the same pasteurella haemolytica bovine immunity test as example 13. 15 animals were assigned to placebo groups receiving only the Miglyol810 excipient, groups receiving 0.5mg/kg,1mg/kg,2mg/kg or 4mg/kg oil dosage forms. Each animal was given a dose of dihydroheptaprenol by subcutaneous injection, and 24 hours later, challenge test was conducted. Observing calf for 10 days, weighing, and performing cadaver test. The judgment is based on the calculated scores of the mortality, the number of calves with the weight gain of 2.27kg during the research period and the lung consolidation. The experiment was designed to have 80% ability to detect the difference.

The mortality rate was: 4 of 15 heads (placebo group), 2 of 15 heads (0.5 and 1mg/kg groups), 0 of 15 heads (2mg/kg group) and 1 of 15 heads (4mg/kg group, with 1 animal dying due to gut torsion, independent of the immunity test). Statistically less dead (P.ltoreq.0.11, unilateral) are those groups which receive 0.5mg/kg or more of dihydroheptaprenyl alcohol. The number of calves with weight gain of more than 2.27kg is: 3 of 15 heads (placebo group), 5 of 15 heads (0.5mg/kg group), 9 of 15 heads (1mg/kg group), 12 of 15 heads (2mg/kg group) and 13 of 15 heads (4mg/kg group). The treatment group receiving 1mg/kg or more of dihydroheptaprenol was statistically different from the placebo group (P.ltoreq.0.03, unilateral). The lung lesion score is: 18.9 ± 8.4% (placebo). 22.7. + -. 16.8% (0.5mg/kg group), 15.6. + -. 14.1% (1mg/kg group), 17.8. + -. 19.9% (2mg/kg group) and 9.3. + -. 10.2% (4mg/kg group). The calf administered 4mg/kg had a significantly lower lung lesion score (P =0.03, unilateral) in the placebo group,

TABLE 1 Whole body protection test for mice

Examples	LD50	ED50mg/kg
			1	28.2	200
2	12	50
			3	10	20
4	22	32
			5	22	32.6
6	22	49.8
			7	22	＜25
8	25	＜12.5
			9	6	25.7
10	9	＜25
			11	25	＜12.5
12	9	79.2

Claims (10)

1. An oil composition for parenteral administration which consists essentially of a dihydropolyisoprene alcohol of formula I and a pharmaceutically acceptable oil.

Wherein n is an integer of 5 to 7.

2. A dihydropolyprenol of the formula i according to claim 1, wherein n = 6.

3. A dihydropolyprenol of the formula I according to claim 1, which is a dihydroheptaprenol.

4. An oil composition according to claim 1 wherein the oil is coconut oil, safflower oil, sesame oil, soybean oil, cottonseed oil, peanut oil or a mineral oil.

5. An oil according to claim 1 which is coconut oil.

6. The oil according to claim 1, which is Miglyol.

7. The oil according to claim 1, which is peanut oil.

8. An oil according to claim 1 which is sesame oil.

9. An oil according to claim 1 which is safflower oil.

10. An oil composition according to claim 1, wherein the dihydropolyprenol of formula i is present in a concentration of from about 1mg/ml to about 500 mg/ml.