AU2000270527A1

AU2000270527A1 - Delayed release compositions and combinations

Info

Publication number: AU2000270527A1
Application number: AU2000270527A
Authority: AU
Inventors: William T. H. Chang
Original assignee: Lytone Enterprise Inc
Current assignee: Lytone Enterprise Inc
Priority date: 2000-08-24
Filing date: 2000-08-24
Publication date: 2002-03-04
Also published as: WO2002015882A1; MXPA03001653A; BR0017324A; CN1434703A; WO2002015882A8

Description

DELAYED RELEASE COMPOSITIONS AND COMBINATIONS

Field of the Invention

The present invention relates to a delayed release composition or combination of a physiologically active protein, which comprises an effective amount of the physiologically active protein, and fibrin glue or fibrinogen and thrombin in an amount effective to delay the release of the physiologically active protein.

Background of the Invention

In the animal husbandry industry, it is believed that the treatment of animals with injection should be limited in order to reduce the stress on the animals, and reduce the labor cost in the meantime. Between the time when pigs are weaned until they are sold, a maximum of two injections is the common practice.

However, to maintain the healthy condition of the raised animals or even promote their growth, the industry might also need to provide some physiologically active proteins to the animals during their growth process. For instance, porcine growth hormone has been reported to be able to stimulate the rate of pig growth for some time. In addition, recombinant porcine somatotropin (rPST) has been shown to be able to reduce the time needed for a pig to reach a market size of approximately 100 kg body weight by more than 10 days. The economic benefit is significant.

However, to achieve the above effect, rPST should be injected to pigs once a day, from the period when the pigs are at 25 kg body weight, until they reach 70 kg body weight. The labor cost involved and the risk of causing stress in pigs are prohibitive for the above commercial practice of rPST.

In considering the method of raising animals, cost, and economic benefit, there is a need to prolong the effective dosage of a physiologically active protein in aiiimals so that only a single injection or treatment would be sufficient to achieve the maximal effect of the physiologically active protein.

Fibrin glue, also known as fibrin sealant, has been utilized in surgery to facilitate coagulation and wound healing for many years. The mechanism of action of fibrin glue is to rnimic the final step of the coagulation cascade wherein fibrinogen is converted to fibrin by the catalization of thrombin. See for example The Journal of Laryngology and Otology November 1987^", Vol. 101. pp. 1182-1186; Bx. J. Surg. 1991, Vol. 78, July, 841-843; and Ann. Thorac Surg 1993, 56:387-9.

Exp Brain Res 1995; 104(2): 199-206 discloses that fibrin glue may prolong the delivery of glial cell line-derived neurotrophic factor (GDNF) to CNS tissues. In addition, Biol. Pharm. Bull. 20(3) 278-281 (1997) describes the site-specific delivery of doxorubicin using a composition of fibrin glue and sodium alginate, which advantageously extends the duration of the drug retained in the tumor extracellular fluid. Furthermore, Cell Transplant 1998 Jan-Feb; 7(1):53-61 teaches a fibrin glue-GDNF slow- release preparation said to be effective for keeping GDNF localized in situ, and protected from circulation and surrounding aqueous humor or CSF.

The above three references, however, do not teach the systemic effect of fibrin glue.

Summary of the Invention

The object of the invention is to provide a method to prolong the effective blood concentration of a physiologically active protein. In particular, the invention provides a delayed release composition and combination of a physiologically active protein. After administration, the physiologically active protein can maintain an effective blood concentration in animals for a prolonged period. Detailed Description of the Invention

It has been surprisingly found that fibrin glue can result in a sustained yet orderly release of physiologically active proteins, and therefore achieve a systemic effect in prolonging the duration of the physiologically active proteins remaining above an effective blood concentration.

Accordingly, the first aspect of the invention provides a delayed release composition of a physiologically active protein, which comprises an effective amount of the physiologically active protein, and fibrin glue in an amount effective to delay the release of the physiologically active protein.

Any protein which is beneficial to economic animals is suitable for use in the invention. Such proteins include, but are not hmited to, growth hormones, insulin, rnterferon and cytokines. The physiologically active protein can be prepared by any method, mcluding genetic engineering recombinant technology. For instance, the physiologically active protein can be recombinant porcine somatotropin (rPST).

The effective amount of the physiologically active protein in the composition of the invention varies along with the species of the physiologically active protein and the effect to be achieved, and can be determined by persons skilled in the art on the basis of ordinary skills in the art. For instance, when rPST is used as the physiologically active protein, the composition of the invention may comprise about 20-50 mg/ml of rPST, preferably about 25 mg/ml of rPST.

Similarly, the amount of fibrin glue effective to delay the release of the physiologically active protein also varies along with the species of the protein and the effect to be achieved, and can be determined by persons skilled in the art on the basis of ordinary skills in the art. In general, the composition of the invention comprises fibrin glue in the amount equivalent to about 30-60 mg/ml of fibrinogen and about 10-100 lU/ml of thrombin. Most preferably, the composition of the invention comprises fibrin glue in the amount equivalent to about 50 mg/ml of fibrinogen and about 60 IU/ml of thrombin.

Thie fibrin glue of the composition according to the invention can be formed in situ at the time of use and achieve the effect of delaying the release of the physiologically active protein. Therefore, the invention in a further aspect provides a delayed release combination of a physiologically active protein which comprises an effective amount of the physiologically active protein, and fibrinogen and thrombin in an amount effective to delay the release of the physiologically active protein.

Typically, the individual components of the delayed release combination of a physiologically active protein according to the invention are contained in separate containers, and are mixed at the injection site at the time of administration to form in situ fibrin glue to delay the release of the physiologically active protein. Alternatively, the physiologically active protein and fibrinogen can be premixed and contained in the same container, and thrombin is contained in a separate container. In this case, the mixture of the physiologically active protein and fibrinogen is mixed with thrombin

^• at the injection site and form in situ fibrin glue to delay the release of the physiologically active protein.

Whether the physiologically active protein is contained in a container alone, or together with fibrinogen, the combination of the invention preferably comprises about 30-60 mg/ml of fibrinogen, and about 10-100

IU/ml of thrombin. Most preferably, the combination of the invention comprises about 50 mg/ml of fibrinogen and about 60 IU/ml of thrombin.

To facilitate the in situ formation of fibrin glue, the delayed release combination of the invention may also comprise a component in group Ila alkaline earth metals, such as an ionized calcium component, e.g. calcium chloride. The delayed release composition or combination of the invention can delay the release of the physiologically active protein contained therein so as to maintain a longer duration of effective blood concentration for the physiologically active protein. Preferably, the delayed release composition or combination of the invention can delay the release of the physiologically active protein so that the protein can maintain an effective blood concentration for at least about 21 days. Most preferably, the effective blood concentration can be maintained for about 30 days.

According to the physiologically active proteins, the delayed release composition or combination of the invention can achieve various effects. For instance, where the physiologically active protein is a growth hormone, the delayed release composition or combination of the invention can be used to promote the growth of economic ariimals, such as pigs, cattle, horses, camels, poultry, aquaculture fish, shrimp, etc., to reduce feed cost and to improve meat quality.

The invention is further detailed by the following examples, which, however, are not limitations of the invention. It should be understood that any variations or modifications which are inferable by persons skilled in the art is contemplated in the invention.

Example 1

30 rats of about 200 gm in weight each were randomly divided into three groups: test group (FG+pST), fibrin glue control group (FG), and daily injection group (DI).

On day 1, the test group rats were injected with lOOμl Solution A containing 10 mg fibrinogen and 1 lOμg pST in 80μl Hanks buffer plus 20μl (100 KIU) bovine lung aprotinin, immediately followed by an injection of lOOμl Solution B containing 40 mM CaCl₂ and 4.9-11.1 mg/ml thrombin at the same injection site. The rats were raised for 21 days, and weighed on day 14 and day 21. On day 1, the fibrin glue control group rats were injected with lOOμl Solution A containing 10 mg fibrinogen in 80 μl Hanks buffer plus 20 μl (100 KIU) bovine lung aprotinin, immediately followed by injection of lOOμl Solution B containing 40 mM CaCl₂ and 4.9-11.1 mg/ml thrombin at the same injection site. The rats were raised for 21 days, and weighed on day 14 and day 21..

The daily injection group rats were injected with 12.5 μl Hanks buffer solution containing 5 μg pST for 21 days. The rats were raised for 21 days, and weighed on day 14 and day 21.

The Hanks buffer solution contains: 5.4 mM KC1, 0.3 mM Na₂HP0₄,

0.4 mM KH₂PO₄, 4.2 mM NaHC0₃, 1.3 mM CaCl₂, 0.5 mM MgCl₂, 0.6 mM MgS0₄, 137 mM NaCl, 5.6 mM D-glucose; and was adjusted to pH

7.4.

The weight-gains in grams at day 14 and day 21 are shown in the following table:

D ay 14 ( g ) D ay 21 ⁽sm)

FG DI FG+pST FG DI FG+pST

20 40 30 35 60 55

25 40 30 50 75 50

50 20 45 65 35 65

40 45 35 60 60 45

45 20 45 55 50 65

35 15 50 55 30 70

30 20 40 50 35 60

10 40 25 25 55 40

45 25 25 60 40 40 30 45

Average 33 30 36 51 49 54 Example 2

Sixteen pigs with an average body weight of 50 kg were selected at random, and separated into 2 groups: a test group and a control group. In the test group, a 2.5 ml Solution A containing 70 mg of porcine somatotropin (Parlow's Laboratory, UCLA Medical Center) and 300 mg porcine plasma fraction contaming about 150 mg fibrinogen in a PBS solution (20mM phosphate, 230 mM NaCl, pH 7) was mixed with a 0.4 ml Solution B containing 2 M CaCl₂ and 500 IU/ml thrombin from bovine serum, and the semi-gel resulting from the combined solutions was injected between the shoulder blades of each pig. In the control group, no pST and fibrin glue were used.

The pigs were fed for a normal commercial diet suitable for pigs at this age. The body weight of each pig was measured every other week for a total period of 7 weeks, and blood samples were taken at the time when the body weight was measured.

The blood samples were analyzed for pH values, urea nitrogen, glucose, and pST concentration. The pigs' loin-eye regions as well as back fat was taken when one half of the control and test pigs were slaughtered at week 3 and the remaining half of the pigs at the conclusion of the trial (week 7). The carcass was also analyzed organoleptically for the meat color, fat tissue, and other meat quality index. The feed efficiency, lean gain, average feed intake, and total body weight were also recorded. Results were statistically analyzed for degree of significance.

Claims

What is claimed is :

1. A delayed release composition of a physiologically active protein, which comprises an effective amount of the physiologically active protein, and fibrin glue in an amount effective to delay the release of the physiologically active protein.

2. The composition according to Claim 1, . wherein the physiologically active protein is selected from the group consisting of growth hormones, insulin, interferon, and cytokines.

3. The composition according to Claim 2, wherein the physiologically active protein is porcine somatotropin.

4. The composition according to Claim 3, wherein the porcine somatotropin is a recombinant porcine somatotropin.

5. The composition according to Claim 4, wherein the effective amount of the recombinant porcine somatotropin is about 20-50 mg/ml.

6. The composition according to Claim 5, wherein the effective amount of the recombinant porcine somatotropin is about 25 mg/ml.

7. The composition according to any of Claims 1-6, . which comprises fibrin glue in the amount equivalent to about 30-60 mg/ml of fibrinogen and about 10-100 IU/ml of thrombin.

8. The composition according to Claim 7, which comprises fibrin glue in the amount equivalent to about 50 mg/ml of fibrinogen and about 60 IU/ml of thrombin.

9. The composition according to Claim 1, which may maintain an effective blood concentration of the physiologically active protein for at least about 21 days.

10. The composition according to Claim 9, which may maintain an effective blood concentration of the physiologically active protein for about 30 days.

11. A delayed release combination of a physiologically active protein, which comprises an effective amount of the physiologically active protein, and fibrinogen and thrombin in an amount effective to delay the release of the physiologically active protein.

12. The combination according to Claim 11, wherein the physiologically active protein is selected from the group consisting of growth hormones, insulin, interferon, and cytokines.

13. The combination according to Claim 12, wherein the physiologically active protein is porcine somatotropin.

14. The combination according to Claim 13, wherein the porcine somatotropin is a recombinant porcine somatotropin.

15. The combination according to Claim 14, wherein the effective amount of the recombinant porcine somatotropin is about 20-50 mg/ml.

16. The combination according to Claim 15, wherein the effective amount of the recombinant porcine somatotropin is about 25 mg/ml.

17. The combination according to any of Claims 11-16, which comprises about 30-60 mg/ml of fibrinogen and about 10-100 IU/ml of thrombin.

18. The combination according to Claim 17, which comprises about 50 mg/ml of fibrinogen and about 60 IU/ml of thrombin.

19. The combination according to any one of Claims 11-16 wherein the physiologically active protein is premixed with the fibrinogen and contained in the same container.

20. The combination according to any one of Claims 11-16, wherein the physiologically active protein and the fibrinogen are contained in separate containers.

21. The combination according to any one of Claims 11-16, which further comprises a component in group Ila a aline earth metals.

22. The combination according to Claim 21, wherein the component in group Ila alkaline earth metals is calcium chloride.

23. The combination according to Claim 11, which may maintain an effective blood concentration of the physiologically active protein for at least about 21 days.

24. The combination according to Claim 23, which may maintain an effective blood concentration of the physiologically active protein for about 30 days.