WO1994023739A1

WO1994023739A1 - METHOD OF DECREASING THE TOXICITY OF THERAPEUTIC COMPOSITIONS USING THYMOSIN β¿4?

Info

Publication number: WO1994023739A1
Application number: PCT/US1994/004359
Authority: WO
Inventors: Allan L. Goldstein; Paul Naylor; Janelle Oliver
Original assignee: The George Washington University
Priority date: 1993-04-22
Filing date: 1994-04-21
Publication date: 1994-10-27
Also published as: AU6771594A

Abstract

A method of substantially inhibiting normal stem cell proliferation in a mammal including the administration of a normal stem cell proliferation-inhibiting effective amount of Tβ4 to the mammal. Also, a method of decreasing the toxicity of a toxic therapeutic composition in a mammal including the administration of a normal stem cell proliferation-inhibiting effective amount of Tβ4 to the mammal.

Description

METHOD OF DECREASING THE TOXICITY OF

THERAPEUTIC COMPOSITIONS USING THYMOSIN β₄

The present invention relates generally to a method of substantially inhibiting normal stem cell proliferation and, more specifically, to a method of decreasing the toxicity of a toxic therapeutic

composition in mammals by administering Thymosin β_A

( "Tβ₄ " ) to said mammals.

Description of Prior Art

Myelopoiesis is the process by which cells in the bone marrow of a mammal mature into several different types of myeloid cells including, but not limited to, granulocytes, monocytes, eosinophils and

megakaryocytes, where these myeloid cells play

important roles in the bodily functions of mammals (e.g., mammals need myeloid cells for proper

functioning of their immune systems). Myelopoiesis typically takes 7 days to be completed. On the first day of myelopoiesis, stem cells in the bone marrow are stimulated to proliferate (i.e., undergo an increased number of mitoses), which results in a "proliferative wave" lasting three days. Within the first few days of myelopoiesis, stem cells proliferate and differentiate to form colony forming cells. The maximum point of cell proliferation is generally reached on the third day of myelopoiesis, at which time the cells in the bone marrow are at the promyelocyte stage. Under normal circumstances, the promyelocyte stage is the latest stage of bone marrow ceil differentiation associated with intense proliferation. Following the third day, maturation generally occurs as bone marrow cells become morphologically recognizable and cell proliferation decreases significantly.

Tβ₄ is a peptide containing 43 amino acids. The amino acid sequence of Tβ₄ is disclosed in U.S. Patent No. 4,297,276, herein incorporated by reference. Tβ₄ was highly conserved during evolution. See, Low et al., "Isolation and Structural Studies of Porcine, Ovine and Murine Thymosin B-4 by High-Performance

Liquid Chromatography," J. Chromatoαr., 301:221 (1984); Spangelo et al., "Biology and Chemistry of Thymosin Peptides: Modulators of Immunity and Neuroendocrine Circuits," Ann. NY Acad. Sci., 496:196 (1987). In fact, total homology exists between murine, rat and human Tβ₄. See, Gondo et al., "Differential Expression of the Human Thymosin B-4 Gene in Lymphocytes,

Macrophages, and Granulocytes," J . Immunol., 139:3840 (1987); Rudin et al., "Differential Splicing of

Thymosin B-4 mRNA, " J. Immunol., 144:4857 (1990);

Wodnar-Filipowicz et al., "Cloning and Sequence

Analysis of cDNA for Rat Spleen Thymosin B-4," Proc. Nat'l Acad. Sci., 81:2295 (1984).

Tβ₄ has been found to be present in numerous tissue types in mammals and has also been implicated in a wide variety of cellular and physiological processes

including inducing terminal deoxynucleotidyl

transferase activity of bone marrow cells, stimulating secretion of hypothalamic luteinizing hormone releasing hormone and luteinizing hormone, inhibiting migration and enhancing antigen presentation of macrophages, and inducing phenotypic changes in T-cell lines in vitro. Recently, it has been suggested that T β₄'s N-terminal tetrapeptide, Ser-Asp-Lys-Pro, inhibits the proliferation of myelopoietic stem cells. Guigon et al., "Inhibition of Human Bone Marrow Progenitors by the Synthetic Tetrapeptide AcSDKP," Exp. Hematol, 18: 1112-15 (1990). Furthermore, it has been suggested that this tetrapeptide ameliorates toxicity induced by the administration of toxic compositions such as cyclophosphamide and cytosine arabinoside ("ara-C"). Bogden, et al., "Amelioration of Chemotherapy Induced Toxicity by Cotreatment with AcSDKP, a Tetrapeptide Inhibitor of Hematopoietic Stem Cell Proliferation," Ann. NY Acad. Sci., 628:126-39 (1991).

Nevertheless, toxicity in mammals, particularly humans, resulting from the administration of toxic therapeutic compositions continues to be a major problem facing the medical profession. Often, the use of effective toxic therapeutic compositions (e.g., the use of AZT to treat AIDS and ara-C to treat cancer) must be restricted or even discontinued because of the toxic side effects which these therapeutic compositions have when they are administered to patients. Thus, even though there are some compounds that appear to be somewhat effective in decreasing toxicity resulting from the administration of a toxic therapeutic

composition to mammals, there remains a need in the art for effective methods of decreasing the toxicity of toxic therapeutic compositions in mammals.

Summary of the Invention

In accordance with the present invention, a method of substantially inhibiting normal stem cell

proliferation in mammals includes administering a normal stem cell proliferation-inhibiting effective amount of Tβ₄ to said mammals.

Further in accordance with the present invention, a method of decreasing the toxicity of a toxic

therapeutic composition in mammals includes

administering a normal stem cell proliferation-inhibiting effective amount of Tβ₄ to said mammals.

Brief Description of the Drawing

Fig. 1 is a graph showing the effect of Tβ₄ and its N-terminal tetrapeptide (N4-Tβ₄) on ara-C treated mice. The ara-C treated mice were injected with a saline control (+-+), 1 μg Tβ₄ (Δ--Δ), 0.1 μg Tβ₄ (0 0), 1 μg

N4-Tβ₄ (Δ●●●Δ) or 0.1 μg N4-Tβ₄ (0●●●0). The timing of administration of ara-C and Tβ₄/N4-Tβ₄ is shown with arrows.

Description of the Preferred Embodiments The term "Tβ₄" as used herein encompasses not only native (i.e., naturally occurring) Tβ₄ but also

synthetic Tβ₄ and recombinant Tβ₄ having the amino acid sequence of native Tβ₄, amino acid sequences

substantially similar thereto, or an abbreviated sequence form thereof, and their analogs and muteins having substituted, deleted, elongated, replaced, or otherwise modified sequences which possess bioactivity substantially similar to that of Tβ₄.

The term "toxic therapeutic composition" as used herein refers to therapeutic agents which, when

administered to a mammal, can be toxic and to mixtures or combinations thereof. Examples of toxic therapeutic compositions include, but are not limited to: anti-infectious agents such as anti-bacterial agents and anti-viral agents, where examples of suitable anti- viral agents include, but are not limited to,

nucleoside analogs (e.g., dideoxynucleosides such as ddl, ddG, ddC, ddA, ddT and analogs thereof,

particularly AZT); anti-cancer agents (e.g., ara-C and methotrexate) and the like.

According to the present invention, methods of substantially inhibiting normal stem cell proliferation are provided. Also provided by the present invention are methods of decreasing the toxicity of a toxic therapeutic composition in mammals. It has been found that these effects can be achieved without

substantially affecting the ability of bone marrow cells to mature normally. The methods of the present invention include the administration of a normal stem ceil proliferation-inhibiting effective amount of Tβ₄ to mammals.

According to a preferred embodiment of the present invention, a normal stem cell proliferation-inhibiting effective amount of Tβ₄ is administered to a subject to substantially inhibit normal stem cell proliferation in the subject. In this embodiment, the subject is preferably a human.

According to another preferred embodiment of the present invention, a normal stem cell proliferation-inhibiting effective amount of Tβ₄ is administered to a mammal subject to decrease the toxicity of a toxic therapeutic composition in the subject. In this embodiment, the subject is preferably a human.

Toxic therapeutic compositions have greater toxicity against rapidly dividing cells such as cancer cells or stem cells which have been stimulated to proliferate by an active agent such as a

chemotherapeutic agent. The administration of a toxic therapeutic composition to such rapidly dividing cells enables the toxic therapeutic composition to harm or perhaps even kill these cells. Accordingly, inhibiting normal stem cell proliferation so that these normal stem cells are not rapidly dividing decreases the toxicity of the toxic therapeutic composition to these cells.

Without being bound to a particular theory, it is believed that normal stem cell proliferation inhibition in a subject results in the substantial inhibition of the cellular processes of the normal stem cells (e.g., cellular uptake of materials and DNA synthesis) in the subject. Under these circumstances, it would be difficult for a toxic therapeutic composition to be toxic to the non-proliferating normal stem cells of the subject because those cells are not contacting or using any extraneous material (i.e., toxic therapeutic compositions) for their cellular processes. Thus, the toxic therapeutic compositions, because they are not contacting or are not being used by non-proliferating normal stem cells, are less toxic to the subject.

For example, the toxicity of AZT results from normal cells incorporating AZT into their DNA

replication process. Such incorporation into the DNA replication process results in incomplete DNA synthesis in those cells (AZT is a "chain terminator") which, in turn, results in toxicity to those cells. Accordingly, where normal stem cells are not proliferating and, therefore, are not synthesizing DNA, nucleoside analogs like AZT cannot be incorporated into the DNA synthesis process and, therefore, cannot be toxic to the normal stem cells.

As can be seen in Table I and the example which follows, Tβ₄ is a substantially more effective normal stem cell proliferation-inhibiting compound than its N- terminal tetrapeptide and is also substantially more effective at decreasing the toxicity of toxic

therapeutic compositions like ara-C in mammals than its N-terminal tetrapeptide.

According to preferred embodiments of the present invention, compositions containing Tβ₄ may be formulated in a conventional manner for administration by any suitable route. Suitable routes of administration include, but are not limited to, oral, rectal, nasal, topical, vaginal, and parenteral (including

subcutaneous, intramuscular, intravenous and

intradermal), with oral or parenteral being preferred. It will be appreciated that the preferred route may vary with the condition, age and species of the

recipient.

While not essential, it is preferable for Tβ₄ to be administered as part of a pharmaceutical formulation. The formulations of the present invention comprise Tβ₄ together with one or more pharmaceutically acceptable carriers and optionally with other therapeutic

ingredients. The carrier((s) are "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.

The formulations include those suitable for oral, rectal, nasal, topical (including buccal and

sublingual), vaginal or parenteral (including

subcutaneous, intramuscular, intravenous and

intradermal) administration. The formulations may conveniently be presented in unit dosage form, e.g., tablets and sustained release capsules, and may be prepared by any suitable pharmaceutical methods.

Such methods include, but are not limited to, the step of bringing into association Tβ₄ with the carrier which constitutes one or more accessory ingredients. In general, the formulations are prepared by uniformly and intimately bringing into association Tβ₄ with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.

Formulations of the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets each containing a predetermined amount of Tβ_4; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in-oil liquid emulsion, etc.

A tablet may be made by compression or molding, optionally with one or more accessory ingredients.

Compressed tablets may be prepared by compressing in a suitable machine Tβ₄ in a free-flowing form such as a powder or granules, optionally mixed with a binder, lubricant, inert diluent, preservative, surface-active or dispersing agent. Molded tablets may be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. The tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein.

Formulations suitable for topical administration include lozenges comprising Tβ₄ in a flavored basis, usually sucrose and acacia or tragacanth; pastilles comprising Tβ₄ in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising Tβ₄ to be administered in a suitable liquid carrier.

Formulations suitable for topical administration to the skin may be presented as ointments, creams, gels and pastes comprising Tβ₄ and a pharmaceutically acceptable carrier. A preferred topical delivery system is a transdermal patch containing the ingredient to be administered.

Formulations for rectal administration may be presented as a suppository with a suitable base

comprising, for example, cocoa butter or a salicylate.

Formulations suitable for nasal administration wherein the carrier is a solid include a coarse powder having a particle size, for example, in the range from about 20 to about 500 microns which is administered in the manner in which snuff is taken, i.e., by rapid inhalation through the nasal passage from a container of the powder held close up to the nose. Suitable formulations wherein the carrier is a liquid, for administration, as for example, a nasal spray or as nasal drops, include aqueous or oily solutions of the active ingredient.

Formulations suitable for vaginal administration may be presented as tampons, creams, gels, pastes, foams or spray formulations containing, in addition to Tβ₄ , suitable carriers.

Formulations suitable for parenteral

administration include aqueous and non-aqueous sterile injection solutions which may optionally contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.

It should be understood that in addition to the ingredients particularly mentioned above the

formulations of this invention may include other

suitable agents having regard to the type of

formulation in question, for example, those suitable for oral administration may include flavoring agents.

A proposed daily dose for administration of the compositions in the present invention is a normal stem cell proliferation-inhibiting effective amount of Tβ₄, which is in a range from about 0.01 to about 2.0 mg of Tβ₄ per kg of body weight of recipient per day

(mg/kg/day), preferably from about 0.02 to about 0.2 mg/kg/day.

In accordance with the present invention, Tβ₄ can be administered in combination with a therapeutically effective amount of a toxic therapeutic composition. Of course, the acceptable dosage range of the toxic therapeutic composition will depend upon the properties of the toxic therapeutic composition (i.e., the

acceptable dosage range will depend upon which toxic therapeutic agent is being administered).

Tβ₄ and a toxic therapeutic composition can be administered "in combination" which, as defined herein, includes various schemes designed to administer Tβ₄ and a toxic therapeutic composition to a subject, whether or not the toxic therapeutic composition and Tβ₄ are administered separately or together, such that the desired dosages of Tβ₄ and the toxic therapeutic

composition are present in the subject at the same time. Any suitable scheme can be used to administer Tβ₄ and a toxic therapeutic composition "in combination" in accordance with the present invention.

The acceptable daily dose of either Tβ₄ alone or Tβ₄ in combination with a toxic therapeutic composition may be conveniently administered in 1 to 3 doses per day. The precise dose administered will depend on the age, condition and species of the recipient.

The invention having been generally described, the following example is given as a particular embodiment of the invention and to demonstrate the practice and advantages thereof. It is understood that the example is given by way of illustration and is not intended to limit the specification or the claims to follow in any manner. Example

Effects of Tβ₄ and N4-Tβ₄ on ara-C Toxicity Tβ₄ and its N-terminal tetrapeptide (N4-Tβ₄) were synthesized by the solid-phase procedure described in Wang et al., "Synthesis of Thymosin β₄ " Int. J. Protein Res., 18:413 (1981). Cytosine arabinoside (ara-C,

Cytosar U-19920, Lot 295AK) was provided as a gift from the Upjohn Company (Kalamazoo, MI) .

C3H/HeN male mice, ages 10-11 weeks, were obtained from Jackson Laboratories. All mice were fed on a standard diet, had free access to water, and were housed in an approved animal care facility according to established guidelines. A total of 160 mice were studied in duplicate experiments, with two equal sets of mice being studied on different days to exclude other variables. Mice were divided into four groups.

Group I received normal saline and Tβ₄ (15 mice). Group II received normal saline and ara-C (29 mice), and served as controls. Group III received ara-C and whole synthetic Tβ₄ (58 mice). Group IV received ara-C and the N-terminai tetrapeptide N4-Tβ₄ (58 mice).

Mice were injected intraperitoneally (i.p.) with 5 mg ara-C in a volume of 0.1 ml sterile Dulbecco's phosphate buffered saline (D-PBS) at 0 , 7 and 36 hours. Thus, a total of 15 mg of ara-C was injected into the mice. The mice were also injected i.p. at 5 and 34 hours with 0.1 ml D-PBS, N4-Tβ₄ in 0.1 ml D-OBS at concentrations of 0.1 μg or 1.0 μg, or Tβ₄ in 0.1 ml D-PBS at concentrations of 0.1 μg or 1.0 μg. Survival was recorded daily. Data from both sets of mice were added to form one larger data set. Calculation of z values using a standard confidence test was used to detect differences in survival between test and control groups.

As the endpoint of the assay was survival and evaluation of short-term toxicity, mice were followed for 2 weeks (16 days), after which the survivors were sacrificed per institutional protocol. Cumulative survival is shown in Table I below and illustrated in

Fig. 1. No decrement in the number of surviving mice was noted between 8 and 16 days. Statistically

significant improvement in survival was noted in groups receiving ara-C plus 0.1 μg Tβ₄ or 0.1 μg N4-Tβ₄ when compared to controls receiving ara-C alone (p < 0.01). This was not seen in groups receiving ara-C plus higher doses of Tβ₄ or N4-Tβ₄. These data suggest a dose-dependent and bifunctional effect of the administered peptides on abrogation of short-term ara-C toxicity. Appreciable side effects were not seen following administration of either peptide, and no deaths were observed in controls receiving doses of 0.1 μg Tβ₄ alone. Both the N-terminal sequence (N4-Tβ₄ and whole Tβ₄ significantly increased survival over controls (p < 0.01). The effect of both peptides was maximal at cumulative doses of 0.2 μg, with less effect at higher doses (p > 0.05 for cumulative doses of 2.0 μg). Such bifunctional activity is commonly seen when cytokine activity is analyzed. More importantly, Tβ₄ was more effective than the N-terminal peptide (p < 0.05), and this effect was demonstrated in the absence of notable side effects.

While the invention has been described and

illustrated with details and references to certain preferred embodiments, those skilled in the art will appreciate that various modifications, changes,

omissions, and substitutes can be made without

departing from the spirit of the invention.

Claims

What is claimed is:

1. A method of substantially inhibiting normal stem cell proliferation in a mammal which comprises administering a normal stem cell proliferation-inhibiting effective amount of Tβ₄ to said mammal.

2. The method of claim 1, wherein said mammal is human.

3. The method of claim 2, wherein the Tβ₄ is administered at a dosage from about 0.01 to about 2.0 mg per kg of body weight per day.

4. The method of claim 2, wherein the Tβ₄ is administered at a dosage from about 0.02 to about 0.2 mg per kg of body weight per day.

5. The method of claim 3, wherein the Tβ₄ is administered parenterally.

6. The method of claim 5, wherein the Tβ₄ is administered intravenously.

7. A composition which comprises a normal stem cell proliferation-inhibiting effective amount of Tβ₄ and a pharmaceutically acceptable carrier.

8. The composition of claim 7, wherein, for each unit dosage, the composition contains from about 0.01 to about 2.0 mg per kg of body weight per day of Tβ₄.

9. The composition of claim 7, wherein, for each unit dosage, the composition contains from about 0.02 to about 0.2 mg per kg of body weight per day of Tβ₄.

10. The composition of claim 8 which is in a form for parenteral administration, wherein the carrier is a sterile liquid carrier suitable for parenteral

administration.

11. The composition of claim 10 which is in a form for intravenous administration.

12. The composition of claim 11, wherein the Tβ₄ is dissolved in a sterile isotonic saline solution.

13. A method for decreasing the toxicity of a toxic therapeutic composition in a mammal which comprises administering to said mammal a normal stem cell proliferation-inhibiting effective amount of Tβ₄.

14. The method of claim 13, wherein said mammal is human.

15. The method of claim 14, wherein the Tβ₄ is administered at a dosage from about 0.01 to about 2.0 mg per kg of body weight per day.

16. The method of claim 14, wherein the Tβ₄ is administered at a dosage from about 0.02 to about 0.2 mg per kg of body weight per day.

17. The method of claim 15, wherein the Tβ₄ is administered parenterally.

18. The method of claim 17, wherein the Tβ₄ is administered intravenously.

19. The method of claim 13, wherein said toxic therapeutic composition is an anti-bacterial agent.

20. The method of claim 13, wherein said toxic therapeutic composition is an anti-viral agent.

21. The method of claim 20, wherein said anti-viral agent is a nucleoside analog.

22. The method of claim 13, wherein said

therapeutic composition is an anti-cancer agent.

23. The method of claim 22, wherein said anti-cancer agent is ara-C.

24. The method of claim 13, wherein said toxic therapeutic composition comprises at least two

therapeutic agents.

25. The method of claim 13, wherein a

therapeutically effective amount of the toxic

therapeutic composition is administered in combination with Tβ₄.

26. The method of claim 25, wherein the toxic therapeutic composition and the Tβ₄ are administered substantially concurrently.