AU2008200190A1 - Method of Administering a Thymosin Alpha 1 Peptide - Google Patents

Description

P/00/011 28/5/91 Regulation 3.2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Name of Applicant: Actual Inventor Address for service is: SciClone Pharmaceuticals, Inc.

Alfred R Rudolph Cynthia W Tuthill WRAY ASSOCIATES Level 4, The Quadrant 1 William Street Perth, WA 6000 Attorney code: WR Invention Title: Method of Administering a Thymosin Alpha 1 Peptide The following statement is a full description of this invention, including the best method of performing it known to me:- 1/1 00 METHOD OF ADMINISTERING A THYMOSIN ALPHA 1 PEPTIDE

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SBACKGROUND OF THE INVENTION ct 1. FIELD OF THE INVENTION The present invention relates to a method of administering a Thymosin C\ 5 alpha 1 peptide.

N 2. DESCRIPTION OF THE BACKGROUND ART 00 CN Thymosin alpha 1 (sometimes referred to as TA1) is a 28- amino acid thymic peptide with immunomodulatory properties, homologous to a natural product originally isolated from thymosin fraction 5 of calf thymus. Its biological effects include augmentation of T lymphocyte function and include modulation of interleukin-2 stimulation of interferon-y production, induction of T lymphocytes and NK cell activity, and stimulation of thymopoiesis. Thymosin alpha 1 also has been shown to up- regulate MHC Class I expression.

Thymosin alpha 1 has previously been suggested for use in certain treatments of cancer, Hepatitis B and C, HIV, etc., e. by subcutaneous injection twice weekly. There remains a need in the art for improved methods of administering Thymosin alpha 1.

SUMMARY OF THE INVENTION In accordance with the present invention, a method of administering a Thymosin alpha 1 (TAI) peptide to a patient in need of immune stimulation, comprises administering the TA1 peptide to the patient so as to substantially continuously maintain an immune stimu!ating-effective amount of the TA1 peptide in the patient during a treatment period of at least about six hours.

In accordance with the present invention there is provided a method of stimulating a patient's immune system with a Thymosin alpha 1 (TA1) peptide -1/2- 00 which comprises continuously infusing a composition consisting essentially of the Thymosin alpha 1 peptide into a patient's circulatory system at a substantially F constant flow rate within a range of about 0.0001-0.1 mg/hr/kg patient body weight, by continuous intravenous infusion so as to substantially continuously maintain an immune stimulating effective amount of said Thymosin alpha 1 peptide in the patient's circulatory system during a treatment period of at least Sabout 6 hours, wherein said continuous infusion impacts a parameter selected from NK activity, level of activated lymphocytes, number of leukocytes, number of granulocytes, number of total lymphocytes, and cytokine expression, such that 00 10 said parameter is greater with said continuous infusion as compared to with subcutaneous injection alone of said Thymosin alpha 1 peptide.

DETAILED DESCRIPTION OF THE INVENTION The present invention is based on a discovery that maintaining immune stimulating-effective amounts of a TA1 peptide in a patient's circulatory system during a treatment period -1/3- 00 provides a substantial improvement in the immune stimulating effect of the TAl peptide.

The invention is applicable to TAl peptides including naturally occurring TAl as well as synthetic TAI and recombinant p TAI having the amino acid sequence of naturally occurring TA1, amino acid sequences substantially similar thereto, or an abbreviated sequence form thereof, and their biologically active S analogs having substituted, deleted, elongated, replaced, or O otherwise modified sequences which possess bioactivity C substantially similar to that of TAI, a TAI derived peptide 00 having sufficient amino acid homology with TAI such that it S functions in substantially the same way with substantially the same activity as TA1.

Because the plasma half-life of subcutaneously injected TAl is only about two hours, according to one embodiment, a TAI peptide such as TAl is administered to a patient in need of immune stimulation so as to substantially continuously maintain an immune stimulating-effective amount of the TAl peptide in the patient's circulatory system during a substantially longer treatment period. Although much longer treatment periods are contemplated in accordance with the present invention, embodiments of the invention include substantially continuously maintaining an immune stimulating-effective amount of the TAI peptide in the patient's circulatory system during treatment periods of at least about.6, 10, 12 hours, or longer. In other embodiments, treatment periods are for at least about a day, and even for a plurality of days, a week or longer. However, it is contemplated that treatments, as defined above, in which immune stimulating-effective amounts of the TAI peptide are substantially continuously maintained in the patient's circulatory system, may be separated by non-treatment periods of similar or different durations.

In accordance with one embodiment, the TAI peptide is continuously infused into a patient, by intravenous infusion, during the treatment period, so as to substantially continuously maintain an immune stimulating-effective amount of the TAt peptide in the patient's circulatory system. The -2- 00 infusion may be carried out by any suitable means, such as by S minipump.

F Alternatively, an injection regimen of the TAl peptide can be maintained so as to substantially continuously maintain an immune stimulating-effective amount of the TAl peptide in the patient's circulatory system. Suitable injection regimens may include an injection every 1, 2, 4, 6, etc. hours, so as to substantially continuously maintain the immune stimulatingeffective amount of the Thymosin alpha 1 peptide in the patient's CN' circulatory system during the treatment period.

00 Although it is contemplated that during continuous S infusion of the TAI peptide, administration will be for a substantially longer duration, according to one embodiment the continuous infusion of the TAl peptide is for a treatment period of at least about 1 hour. More preferably, continuous infusion is carried out for longer periods, such as for periods of at least about 6, 8, 10, 12 hours, or longer. In other embodiments, continuous infusion is for at least about one day, and even for a plurality of days such as for one week or more.

Immune stimulating-effective amounts of a TAl peptide may be substantially continuously maintained in a patient's circulatory system by administering the TAl peptide to the patient at a rate within a range of about 0.0001-0.1 mg/hr/Kg patient body weight. Preferred administration rates are within a range of about 0.0003-0.03 mg/hr/Kg patient body weight.

In preferred embodiments, the TAl peptide is present in a pharmaceutically acceptable liquid carrier, such as water for injection, saline in physiological concentrations, or similar.

The invention may be utilized for treatment of any patient in need of immune stimulation, including cancer patients, HIV patients, and patients having various forms of hepatitis, including Hepatitis B and Hepatitis C. For example, the invention may be utilized to promote bone marrow recovery in cancer patients following chemotherapy. The invention may be particularly useful for addition of TAl to chemoimmunotherapy for increased survival in melanoma and hepatocellular carcinoma (HCC) 00 patients, and for reduction of haematological toxicity in lung O cancer.

In the following examples, which are not intended to be limiting, a continuous infusion of TA1 was evaluated in a cancer therapy model with the use of surgically implanted osmotic minipumps, which deliver fluids at a constant flow rate for days. Rats were given 5-fluorouracil (5-FU) to cause immune CO suppression, and then treated with injected or infused TA1 8 days later (the nadir of white cell count after 5-FU). Treatment -q groups, 8 rats each, were: control (minipumps with saline); low 0 dose TA1 (0.2 mg/Kg sc injection; empty minipumps); high dose TA1 S (3.5 mg/Kg sc injection; empty minipumps); and high dose infused TA1 (3.5 mg/Kg infused by minipumps). Immune parameters were determined at baseline and 8 days after 5-FU treatment (day 1 of STA1 treatment), and also at 5, 12, 20, and 27 days after TA1 treatment.

Example 1 week old rats, weighing 250 300 g, received 100 mg/kg 5-fluorouracil (5-FU) for immune suppression.

8 days after 5-FU treatment, rats were randomly assigned to one of the following groups SControl (saline in minipump) SLow dose TA1, injected s.c. at 0.2 mg/Kg (with empty minipumps) High dose TA1, injected s.c. at 3.5 mg/Kg (with empty minipumps) Continuous infusion TA1, provided by minipump at 3.5 days Immune parameters were determined at baseline and 8 days after 5-FU treatment (day 1 of TA1 treatment), and also at 5, 12, and 27 days after TA1 treatment.

The evaluations included NK activity (LDH released from YAC- 1 cells after 4h exposure to PBMC), total leukocyte number (judged by physical cytofluorimetric parameters, after verifying 00 the specificity by monoclonal antibody), total lymphocyte number (CD3+ by flow cytometry), and activated lymphocytes (CD25+CD3+ by C N flow cytometry).

SNK activity was 42 5 at baseline and was depressed to 9 2 after 5-FU. Low dose TA1 treatment lead to a significant recovery of NK activity after 12 days, while high dose TA1 achieved significant recovery in only 5 days. Continuous infusion S of TA1, however, was able to double the response at 5 days, to 32 4% (versus 16 2 for high dose injected, 12 3 low d6se S0 injected, and 11 1 control). Only animals treated with TA1 by 00 0 continuous infusion had a complete recovery of NK activity to baseline levels.

Total white blood cell count, as determined by morphology, was depressed from 14,590 2,071 cells/mm 3 to 2,597 582 after Streatment with 5-FU. Low or high dose TA1 treatment by injection trended towards a sooner increase in recovery compared to untreated animals. Continuous infusion of TA1, however, provided statistically significant and complete recovery to baseline levels after only 5 days.

Activated lymphocytes (CD3+CD25+) were not decreased significantly by 5-FU treatment (from 65 21 cells/mm 3 to 37 however, the levels were dramatically increased 12 and days after high dose TA1 treatment (297 136 and 321 cells/mm 3 vs 166 70 and 212 77 cells/mm 3 respectively). TA1 provided by continuous infusion lead to an even greater increase, to 422 105 and 446 73 cells/mm 3 Example 2 week old rats, weighing 250 300 g, received 100 pg/kg for immune suppression.

8 days after 5-FU treatment, rats were randomly assigned to one of the following groups S Control (saline in minipump) S High dose TA1, injected s.c. at 3.5 mg/Kg (with empty minipumps) Continuous infusion TA1, provided by minipump at days 00 Immune parameters were determined at baseline and 8 days C after 5-FU treatment (day 1 of TA1 treatment), and also at 5 and 14 days after TA1 treatment.

SThe evaluations included total leukocyte number (judged by physical cytofluorimetric parameters, after verifying the specificity by monoclonal antibody), granulocytes (flow cytometry using FITC anti rat granulocyte HIS-48), total lymphocyte number _0 (CD3+ by flow cytometry), T helper lymphocytes (CD4+ by flow cytometry), activated lymphocytes (CD25+CD3+ by flow cytometry), C0 and cytokine expression in plasma (IL-2 and IFN-y by ELISA).

00 SAfter determining in Example 1 that TAl provided by C continuous infusion compared to s.c. injection had a dramatic effect on the total number of leukocytes, it was of interest to determine which type of white blood cell was responsible for the increase. Granulocytes appear to be the subset of white blood cells that are most affected by TAl provided by continuous infusion. The number of granulocytes was decreased after from 4,485 1,116 to 1,249 432. Treatment with TA1 resulted in an increase to 14,652 2,463 within 5 days (compared to 9,924 3,218 with TAl by injection or 6,954 1,519 with no TA1), and this level was still the highest after 14 days.

Interestingly, there was one animal in this study which was provided TA1 by BOTH injection (of 3.5 mg/Kg) and by continuous infusion (of another 3.5 mg/Kg). Not only was this animal healthy and vigorous, with no obvious adverse events, but the TA1 effects on the immune parameters measured were even greater than those in the other animals. For granulocytes, this study animal had a greatly increased level of 19,376 cells/mm 3 after 5 days, compared to the mean of 14,652 2,463 in the other infused animals.

The number of total lymphocytes (CD3+) was dramatically decreased by 5-FU treatment (from 10,904 1,973 cells/mm 3 to 1,740 560). Treatment with TAl allowed for a recovery to baseline levels, which occurred after only 5 days when TAl was provided by continuous infusion but was not seen until 14 days for injected TA1.

00 The animal that had TA1 provided by both injection and infusion had levels of lymphocytes which were not much different from the other animals (9,765 cells/mm 3 compared to the mean of 9,644 961), but the percentage of these lymphocytes which were 5 activated was greatly increased (from 428 89, or 4% of lymphocytes, for the animals with TAl by infusion, to 976, or of lymphocytes, for the animal which had TA1 in a high dose S injection followed by infusion).

0 T helper lymphocytes (CD3+CD4+) were also depressed by C- treatment with 5-FU, from 5,411 1,084 cells/mm 3 to 1,710 00 0 449. These depressed levels of T cells did not increase without C, treatment with TA1 for the 14 days of the experiment. By contrast with the results seen for granulocytes, in which TA1 provided by continuous infusion was superior to TA1 provided by Sinjection for recovery of cell numbers, TA1 provided by either delivery method was sufficient to return the levels of T helper cells to baseline.

Since TAl provided either by injection or by continuous infusion lead to an increase in CD4+ T helper lymphocytes, it was of interest to determine whether this increase was due to an effect on the Thl or the Th2 subset of T helper cells. Previous in vitro and in vivo data have demonstrated that TA1 increases the Thl subset of T cells, and in this study the same effect was seen. Providing TA1 by continuous infusion lead to an even greater increase in the plasma level of the Thl cytokine IL-2 than was seen after s.c. injection (42 7 pg/ml 14 days after TA1 by continuous infusion, compared to 21 16 for injected TA1 and 10 16 for control animals).

Treatment by TAl lead to an increase in the Thl cytokine IL- 2, and TA1 allows for an increase in another Thl cytokine, IFNy. Although the levels are low, by 5 days after treatment, s.c.

injected TAl lead to higher plasma levels of IFN-y. By 14 days after treatment the animals with TA1 provided by continuous infusion had the highest levels (14 5 pg/ml compared to 10 1 by injection or 8 8 for control).

The animal which received TA1 by both injection and continuous infusion had even greater levels of both of the Thl cytokines measured, especially IFN-y, which was pg/ml after 14 days, compared to 14 5 pg/ml for the other animals.

O CONCLUSIONS: Maintenance of a constant level of TAI over a plurality

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00 of days in the circulation increases the measured Simmunological effects.

SThis dosage regimen leads to unexpected positive effects on granulocytes, as well as the positive effects on monocytes seen after injection of TA1.

SNo adverse events were observed, even at doses of TAI times higher than usual (and in one animal, at doses 30 times higher than usual).

Throughout the specification and claims, unless the context requires otherwise, the word "comprise" or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.

Each document, reference, patent application or patent cited in this text is expressly incorporated herein in their entirety by reference, which means that it should be read and considered by the reader as part of this text. That the document, reference, patent application or patent cited in this text is not repeated in this text is merely for reasons of conciseness.

-8/1- 00 Reference to cited material or information contained in EI the text should not be understood as a concession that ,t the material or information was part of the common general knowledge or was known in Australia or any other country.

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Claims (11)

1. 2. The method of claim 1 wherein said treatment period is at least about 8 hours.
2. 3. The method of claim I wherein said treatment period is at least about hours.
3. 4. The method of claim I wherein said treatment period is at least about 12 hours. The method of claim 1 wherein said treatment period is at least about one day.
4. 6. The method of claim 1 wherein said treatment period is about one week or more.
5. 7. The method of claim 1 wherein said Thymosin alpha 1 peptide is present in a pharmaceutically acceptable liquid carrier. -9-
6. 8. The method of claim 1 wherein said Thymosin alpha 1 peptide is oo 0 administered to said patient at a rate of about 0.0003-0.03 mg/hr/kg patient body C weight.
7. 9. The method of claim 1 wherein said intravenous infusion is carried out by minipump. The method of claim 9 wherein said minipump is an osmotic minipump. S11. The method of claim 10 wherein said minipump is implanted in said patient. 00 S12. The method of claim 1 wherein said patient is a cancer patient.
8. 13. The method of claim 1 wherein said cytokine is a Thl cytokine,
9. 14. The method of claim 1 wherein said cytokine is IL-2. The method of claim 1 wherein said cytokine is IFN-y.
10. 16. The method of claim 1 wherein said Thymosin alpha 1 peptide has substantially an amino acid sequence of naturally occurring Thymosin alpha 1.
11. 17. The method of claim 1 substantially as herein before described with reference to the Examples.