WO2006091661A1

WO2006091661A1 - Compositions for and methods of modulating platelet levels

Info

Publication number: WO2006091661A1
Application number: PCT/US2006/006261
Authority: WO
Inventors: David B. Weiner
Original assignee: The Trustees Of The University Of Pennsylvania
Priority date: 2005-02-22
Filing date: 2006-02-22
Publication date: 2006-08-31

Abstract

Compositions for and methods of treating individuals who have elevated platelet levels are disclosed. The methods comprise the step of administering to the individual a therapeutically effective amount Vpr protein; a function fragment of Vpr protein; a nucleic acid encoding Vpr protein operably linked to regulatory elements; or a nucleic acid encoding a functional fragment of Vpr protein operably linked to regulatory elements. Compositions for and methods of treating individuals who have insufficient platelet levels are disclosed. The methods comprising the step of administering to the individual an amount of one or more glucocorticoid receptor antagonist compounds effective to increase platelet number.

Description

COMPOSITIONS FOR AND METHODS OF MODULATING PLATELET LEVELS

FIELD OF THE INVENTION

The present invention relates to methods of treating individuals who have diseases and conditions which affect platelet levels, and to pharmaceutical compositions useful in such methods.

BACKGROUND OF THE INVENTION

Essential thrombocythaemia is defined as an increase in the number of platelets. The condition is due to an increase or overproduction of platelets by the bone marrow, although the underlying cause is unknown. It is rare, affecting approximately one person per 100,000.

In addition to Essential thrombocythaemia, elevated platelet levels are observed in individuals with chronic myeloid leukaemia, Polycythaemia vera, Myelofibrosis, and Myelodysplastic syndromes. Further, numerous individuals experience secondary or reactive thrombocytosis. Such individuals may have acute and/or chronic infection, inflammatory disorders such as, for example, Kawasaki's disease, chronic iron deficiency, acute or chronic blood loss, tissue damage from trauma or surgery, reactions to certain medicines, such as, for example, conditions following splenectomy and hyposplenism, Malignancy (Hodgkin's disease, solid tumours), Rebound from chemotherapy,

The elevated number of platelets increases the risk of bleeding and/or the formation of blood clots (thrombosis). The outcome for sufferers is variable, ranging from prolonged periods without complications in some people, to complications related to haemorrhage and thrombosis in others.

Thrombocytopenia, which is any disorder in which there are insufficient platelets, is generally divided into three major causes of low platelets: low production of platelets in the bone marrow, increased breakdown of platelets in the bloodstream (called intravascular), increased breakdown of platelets in the spleen or liver (called extravascular). Disorders that involve low production in the bone marrow include HIV infection where there is an observed decrease of platelets associated with progression of infection ands disease. Thrombocytopenia is sometimes associated with abnormal bleeding. There remains a need for methods and pharmaceutical compositions to treat these various diseases, disorders and conditions characterized by elevated or depressed levels of platelets.

SUMMARY OF THE INVENTION

The present invention relates to methods of treating an individual who has been diagnosed as having elevated platelet levels. The methods comprise the step of administering to the individual, in an amount effective to decrease platelet numbers, a pharmaceutical composition comprising one or more of the components selected from the group consisting of: Vpr protein; a function fragment of Vpr protein; a nucleic acid encoding Vpr protein operably linked to regulatory elements; and a nucleic acid encoding a functional fragment of Vpr protein operably linked to regulatory elements.

The present invention also relates to compositions for treating an individual who has been diagnosed as having elevated platelet levels. The compositions comprise, in an amount effective to decrease platelet numbers, one or more of the components selected from the group consisting of: Vpr protein; a function fragment of Vpr protein; a nucleic acid encoding Vpr protein operably linked to regulatory elements; and a nucleic acid encoding a functional fragment of Vpr protein operably linked to regulatory elements.

The present invention further relates to methods of treating an individual who has been diagnosed as having insufficient platelet levels. The methods comprise the step of administering to the individual, in an amount effective to increase platelet numbers, one or more glucocorticoid receptor antagonist compounds.

The present invention also relates to compositions for treating an individual who has been diagnosed as having insufficient platelet levels. The compositions comprise, in an amount effective to increase platelet numbers, one or more glucocorticoid receptor antagonist compounds. DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention provides improved methods to modulating platelet numbers in an individual. According to the present invention, the HIV protein Vpr suppresses platelet numbers and that glucocorticoid receptor antagonists elevate platelet numbers such is in HIV infected individuals who have decreased platelet numbers. While not limiting the invention to any one mechanism, Vpr is involved in a glucocorticoid pathway which is inhibited by glucocorticoid receptor antagonists. Progression of infection corresponding to increasing Vpr levels also corresponds to a decrease in platelet numbers. Inhibition of infection with a glucocorticoid receptor antagonist leads to an increasing in platelets.

SIV-infected macaques were treated with mifepristone, a steroidal based glucocorticoid receptor antagonist. In addition to a decrease in viral titers, mifepristone treated SIV positive macaques were observed to have an increase in platelet number. SIV positive macaques treated with other antiviral compounds also had lower viral load. However, the other anti-virals had no effect on platelet count. SIV infected macaques and HIV infected humans who are experiencing AIDS symptoms have low platelet counts. Reversing the decline in platelet number is a positive affect of mifepristone treatment.

Vpr inhibits megakaryocytes from making platelets. Vpr inhibits platelet production and mifepristone reverses Vpr's effect.

According to the invention, Vpr can be used to treat conditions associated with elevated platelet counts and glucocorticoid receptor antagonist, particularly steroidal based compounds such as mifepristone may be useful to treat conditions associated with depressed platelet counts if the platelet counts are low because of some interference with platelet production involving the pathway that Vpr acts upon, such as HTV infected individuals with depressed platelet counts.

The amino acid sequence of Vpr and the DNA sequence that encodes it are described in U.S. Patent No. 5,874,225 issued on February 23, 1999, which is incorporated herein by reference, including the patents and publications referred to therein. Fragments of Vpr are described in PCT/US94/02191 filed February 22, 1994, PCT/US95/12344 filed September 21, 1995, and PCT/US98/16890 filed August 14, 1998, which are each incorporated herein by reference together with the respective corresponding U.S. National Stage applications claiming priority thereto, and U.S. Patent No. 5,763,190 issued June 9, 1998, which is incorporated herein by reference. U.S. Serial 08/167,608 filed December 15, 1993 and PCT/US94/00899 filed January 26, 1994, which are incorporated herein by reference, describe recombinant viral particles which include functional fragments of Vpr protein as part of the viral particle. Mutational analysis of the Vpr protein has identified distinct functional domains required for the nuclear localization and cell cycle arrest activity of Vpr (Mahalingam, S, et al, 1997, J. Virol., 71, 6339-6347; Macreadie, LG. et al, 1995, Proc. Natl. Acad. ScL, 92, 2770-2774; both of which are incorporated by reference herein).

The present invention provides a method to treat individuals who have been identified as having a condition selected from the group consisting of: Essential thrombocythaemia, chronic myeloid leukaemia, Polycythaemia vera, Myelofibrosis, and Myelodysplastic syndromes. In treating such individuals, platelet levels are increased. The present invention also provides methods of treating individuals who have been identified as having secondary or reactive thrombocytosis such as those having a condition selected from the group consisting of: acute and/or chronic infection, inflammatory disorders such as for example, chronic iron deficiency, acute or chronic blood loss, tissue damage from trauma or surgery, side effects of medicine, such as steroid, or vincristine, splenectomy and hyposplenism, malignancy, such as Hodgkin's disease or solid tumours, and those rebounding from chemotherapy. In treating such individuals, platelet levels are increased.

According to the invention, methods comprise administering to the individual amount of Vpr protein, as Vpr protein or a functional fragment thereof, or as a nucleic acid encoding Vpr protein or a functional fragment thereof, or a combination of two or more of the same effective to decrease platelet number. When a nucleic acid encoding Vpr protein or a functional fragment thereof is delivered to an individual, the coding sequence is operably linked to regulatory elements. Once delivered to the individual, the nucleic acid encoding the Vpr protein is expressed and the Vpr protein is synthesized within the individual. In some embodiments, the Vpr is delivered as a nucleic acid molecule with the coding sequence for Vpr protein and/or a functional fragment thereof. In some embodiments, the Vpr and/or a functional fragment thereof is delivered as a protein. Once delivered to the individual, the presence of the Vpr protein, either delivered as a protein or produced by the expression of the nucleic acid molecule that encodes it, inhibits platelet production.

An effective daily dosage of Vpr protein and/or functional fragments thereof can be about 0.1 to 100 milligrams per kilogram of body weight. Ordinarily 0.5 to 50, and preferably 1 to 10 milligrams per kilogram body weight per day. Nucleic acids encoding Vpr protein and/or functional fragments thereof can be coadministered as described above. An effective dose of the nucleic acid encoding the Vpr protein and/or functional fragments thereof is a dose of about Ing to lOmg of nucleic acid; in some embodiments, about 0.1 to about 2000 micrograms of DNA. In some preferred embodiments, therapeutically active dose is about 1 to about 1000 micrograms of DNA. In some preferred embodiments, therapeutically active dose is about 1 to about 500 micrograms of DNA. In some preferred embodiments, therapeutically active dose is about 25 to about 250 micrograms of DNA. Most preferably, therapeutically active dose is about 100 micrograms DNA. The dosage regimen for treating an individual may comprise administering one or more nucleic acid encoding the Vpr protein and/or functional fragments thereof, and Vpr protein and/or functional fragments thereof in one or more doses. The daily dosage of Vpr maybe given in divided doses 1 to 6 times a day or in sustained release form is effective to obtain desired results. The administration of Vpr proteins and/or functional fragments thereof, and/or nucleic acids that encode Vpr and/or functional fragments thereof may be repeated one to six times a day, for one to several days or weeks, or until platelet levels have reached a desirable range. In preferred embodiment, the Vpr protein and/or functional fragments thereof, and/or nucleic acids encoding them are administered by continuous infusion of the Vpr protein. In another preferred embodiment, the Vpr protein and/or functional fragments thereof, and/or nucleic acids encoding them are administered in a bolus dose, followed by continuous infusion of the Vpr protein and/or functional fragments thereof, and/or nucleic acids encoding them.

In some embodiments of the preventive and therapeutic methods of the present invention, a combination of one or more of Vpr, a functional fragment thereof, a nucleic acid encoding Vpr, or a nucleic acid encoding a functional fragment of Vpr is administered to a patient. In some embodiments, the Vpr or a functional fragment thereof is administered to the individual in the same formulation as the nucleic acid encoding Vpr. In other embodiments, the Vpr or a functional fragment thereof is administered to the individual in a separate formulation than the nucleic acid encoding Vpr.

Compositions and methods for delivering proteins, such as Vpr, to cells by direct DNA administration have been reported using a variety of protocols. Examples of such methods are described in U.S. Patent No. 5,593,972, U.S. Patent No. 5,739,118, U.S. Patent No. 5,580,859, U.S. Patent No. 5,589,466, U.S. Patent No. 5,703,055, U.S. Patent No. 5,622,712, U.S. Patent No. 5,459,127, U.S. Patent No. 5,676,954, U.S. Patent No. 5,614,503, and PCT Application PCT/US95/12502, which are each incorporated herein by reference. Compositions and methods for delivering proteins to cells by direct DNA administration are also described in PCT/US90/01515, PCT/US93/02338, PCT/US93/048131, and PCT/US94/00899, which are each incorporated herein by reference. In addition to the delivery protocols described in those applications, alternative methods of delivering DNA are described in U.S. Patent Nos. 4,945,050 and 5,036,006, which are both incorporated herein by reference. Nucleic acid molecules can also be delivered using liposome-mediated DNA transfer such as that which is described in U.S. Patent No. 4,235,871, U.S. Patent No. 4,241,046 and U.S. Patent No. 4,394,448, which are each incorporated herein by reference.

Formulations comprising the nucleic acid having a sequence encoding Vpr are made up according to the mode and site of administration. Such formulation is well within the skill in the art. In addition to nucleic acids and optionally polypeptides, the formulation may also include buffers, excipients, stabilizers, carriers and diluents.

The pharmaceutical composition comprising Vpr protein or a fragment thereof and a pharmaceutically acceptable carrier or diluent may be formulated by one having ordinary skill in the art with compositions selected depending upon the chosen mode of administration. Suitable pharmaceutical carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field which is incorporated herein by reference. For parenteral administration, the Vpr protein or fragments thereof can be, for example, formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils may also be used. The vehicle or lyophilized powder may contain additives that maintain isotonicity (e.g.,¹ sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The foπnulation is sterilized by commonly used techniques. For example, a parenteral composition suitable for administration by injection is prepared by dissolving 1.5% by weight of active ingredient in 0.9% sodium chloride solution.

The pharmaceutical compositions comprising Vpr protein, or fragments thereof may be administered by any means that enables the active agent to reach the agent's site of action in the body of a mammal. Because proteins are subject to being digested when administered orally, parenteral administration, i.e., intravenous, subcutaneous, intramuscular, would ordinarily be used to optimize absorption.

The dosage administered varies depending upon factors such as: pharmacodynamic characteristics; its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms; kind of concurrent treatment; and frequency of treatment. Usually, a daily dosage of Vpr protein can be about 0.1 to 100 milligrams per kilogram of body weight. Ordinarily 0.5 to 50, and preferably 1 to 10 milligrams per kilogram per day given in divided doses 1 to 6 times a day or in sustained release form or continuous administration is effective to obtain desired results.

Another aspect of the present invention relates to pharmaceutical compositions that comprise a nucleic acid molecule that encodes Vpr and a pharmaceutically acceptable carrier or diluent. According to the present invention, genetic material that encodes Vpr protein is delivered to an individual in an expressible form. The genetic material, DNA or RNA, is taken up by the cells of the individual and expressed. Vpr that is thereby produced can inhibit immune responses, either those directed at an immunogenic vector or another undesirable immune response such as those associated with autoimmune and inflammatory disease and conditions and transplantation procedures. Thus, pharmaceutical compositions comprising genetic material that encodes Vpr are useful in the same manner as pharmaceutical compositions comprising Vpr protein.

Nucleotide sequences that encode Vpr protein operably linked to regulatory elements necessary for expression in the individual's cell may be delivered as pharmaceutical compositions using a number of strategies which include, but are not limited to, either viral vectors such as adenovirus or retrovirus vectors or direct nucleic acid transfer. Methods of delivery nucleic acids encoding proteins of interest using viral vectors are widely reported. A recombinant viral vector such as a retrovirus vector or adenovirus vector is prepared using routine methods and starting materials. The recombinant viral vector comprises a nucleotide sequence that encodes Vpr. Such a vector is combined with a pharmaceutically acceptable carrier or diluent. The resulting pharmaceutical preparation may be administered to an individual. Once an individual is infected with the viral vector, Vpr is produced in the infected cells.

Alternatively, a molecule which comprises a nucleotide sequence that encodes Vpr can be administered as a pharmaceutical composition without the use of infectious vectors. The nucleic acid molecule may be DNA or RNA, preferably DNA. The DNA molecule may be linear or circular, it is preferably a plasmid. The nucleic acid molecule is combined with a pharmaceutically acceptable carrier or diluent.

According to the invention, the pharmaceutical composition comprising a nucleic acid sequence that encodes Vpr protein may be administered directly into the individual or delivered ex vivo into removed cells of the individual which are reimplanted after administration. By either route, the genetic material is introduced into cells which are present in the body of the individual. Preferred routes of administration include intramuscular, intraperitoneal, intradermal and subcutaneous injection. Alternatively, the pharmaceutical composition may be introduced by various means into cells that are removed from the individual. Such means include, for example, transfection, electroporation and microprojectile bombardment. After the nucleic acid molecule is taken up by the cells, they are reimplanted into the individual.

The pharmaceutical compositions according to this aspect of the present invention comprise about Ing to lOmg of nucleic acid in the formulation; in some embodiments, about 0.1 to about 2000 micrograms of DNA. In some preferred embodiments, the pharmaceutical compositions contain about 1 to about 1000 micrograms of DNA. In some preferred embodiments, the pharmaceutical compositions contain about 1 to about 500 micrograms of DNA. In some preferred embodiments, the pharmaceutical compositions contain about 25 to about 250 micrograms of DNA. Most preferably, the pharmaceutical compositions contain about 100 micrograms DNA.

The pharmaceutical compositions according to this aspect of the present invention are formulated according to the mode of administration to be used. One having ordinary skill in the art can readily formulate a nucleic acid molecule that encodes Vpr. In cases where injection is the chosen mode of administration, a sterile, isotonic, non-pyrogenic formulation is used. Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose. Isotonic solutions such as phosphate buffered saline are preferred. Stabilizers include gelatin and albumin.

Regulatory elements for nucleic acid expression include promoters, initiation codons, stop codons, and polyadenylation signals. It is necessary that these regulatory elements be operably linked to the sequence that encodes the desired polypeptides and optionally the Vpr polypeptide and that the regulatory elements are operable in the individual to whom the nucleic acids are administered. For example, the initiation and teπnination codons must be in frame with the coding sequence. Promoters and polyadenylation signals used must also be functional within the cells of the individual.

Examples of promoters useful to practice the present invention include but are not limited to promoters from Simian Virus 40 (SV40), Mouse Mammary Tumor Virus (MMTV) promoter, Human Immunodeficiency Virus (HIV) such as the HIV Long Terminal Repeat (LTR) promoter, Moloney virus, ALV, Cytomegalovirus (CMV) such as the CMV immediate early promoter, Epstein Barr Virus (EBV), Rous Sarcoma Virus (RSV) as well as promoters from human genes such as human Actin, human Myosin, human Hemoglobin, human muscle creatine and human metallothionein. Examples of polyadenylation signals useful to practice the present invention include but are not limited to SV40 polyadenylation signals and LTR polyadenylation signals. In particular, the SV40 polyadenylation signal which is in pCEP4 plasmid (Invitrogen, San Diego CA), referred to as the SV40 polyadenylation signal, is used.

Vpr levels increase as HTV infection progresses. Platelet counts become depressed as HIV infection proceeds to AIDS. GRII antagonist drugs, including Mifepristone, which can be used to inhibit HIV replication, can be used to increase platelet levels in HIV infected individuals and may also be useful in the treatment of other diseases, disorders or conditions which are characterized by suppressed platelet number. The compounds useful in the invention may act as steroid hormone receptor antagonists. According to the present invention, glucocorticoid receptor antagonist compounds may be used to treat individuals with suppressed platelet counts such as HIV infected individuals, or those suffering from some other forms of thrombocytopenia. Thrombocytopenia is observed in cases of Aplastic anemia, cancer and infections in bone marrow, and as a side effect to some drugs, as well as in disorders involving platelet breakdown such as Immune or Thrombotic thrombocytopenic purpura; Drug-induced thrombocytopenia (immune and nonimmune), primary thrombocythemia, disseminated intravascular coagulation and hypersplenism. Amounts of such compounds effective to increase platelet counts are administered to such individuals.

Examples of compounds useful in the present invention include glucocorticoid receptor type II antagonist compounds, particularly those having a steroidal structure. In accordance with the present invention, a preferred group of glucocorticoid receptor antagonists are those to which mifepristone belongs. This compound, 1 lβ-(4-dimethylaminophenyl)-17β.-hydroxy-17β.-(propyl-l-ynyl )estra- 4,9-dien-3-one, is a good glucocorticoid antagonist, which also has antiprogestin activity. Further details concerning this and related compounds may be found in Agarwal, M. K. et al. "Glucocorticoid antagonists" FEBS LETTERS 217, 221-226 (1987), which is incorporated herein by reference. As noted, agonist activity requires an alcohol group at the 1 lβ position. Accordingly, glucocorticoid receptor antagonists used in the present invention preferably are not hydroxylated at the 1 lβ position. In preferred embodiments, the glucocorticoid receptor antagonists used in the present invention preferably are not hydroxylated at the 1 lβ position and are resistant to reactions in which the 1 lβ position becomes hydroxylated. Extensive work has been done over the years in synthesizing and testing glucocorticoid antagonists which belong to this group, and the published literature is an abundant guide for the selection of candidate compounds that fall within the scope of the present invention. The patent literature alone is substantial. Thus, reference is made to the following U.S. patents, all of which are incorporated herein by reference in their entirety, and particularly with respect to the descriptions of the compounds disclosed therein being described herein and their use in the treatment of individuals infected with virus as well as individuals suffering from viral disease, and the prevention of viral infection in individuals exposed to a virus: U.S. Pat. Nos. 4,296,206; 4,386,085; 4,447,424; 4,477,445; 4,519,946; 4,540,686; 4,547,493; 4,634,695; 4,634,696; 4,753,932; 4,774,236; 4,814,327; 4,829,060; 4,861,763; 4,912,097; 4,943,566; 4,954,490; 4,978,657; 5,006,518; 5,043,332; 5,064,822; 5,073,548; 5,089,488; 5,089,635; 5,093,507; 5,095,010; 5,095,129; 5,132,299; 5,166,146; and 5,276,023. Analysis of the patents set out above and the attendant technical literature reveals that the 11 -position substituent, and particularly the size of that substituent, plays a key role in determining the antiglucocorticoid activity. The character of the A ring is also important. It is also noted that a 17-hydroxypropenyl side chain generally decreases antiglucocorticoidal activity in comparison to the 17-propinyl side chain containing compounds, and that generally 9α, 10α-CH2 groups decrease antiglucocorticoidal activity. In some embodiments, the compounds described in GB 929271 and U.S. Patent No. 3,362,968, which are each incorporated herein by reference, are used. In some embodiments, the compounds described in U.S. Patent No. 3,024,256, which is incorporated herein by reference, are used. In some embodiments, the compounds described in Indian patent IN 33649 and IN 67932 and PCT publication WO92/19616, which are each incorporated herein by reference are used. In some embodiments, the compounds described in JP 62012791 and WOO 1/01996, which are each incorporated herein by reference, are utilized. The pharmaceutical compositions used in the present invention may be administered by any means that enables the active agent to reach the agent's site of action in the body of the individual. Pharmaceutical compositions of the present invention may be administered by conventional routes of pharmaceutical administration. Pharmaceutical compositions may be administered parenterally, i.e. intravenous, subcutaneous, intramuscular, subdermally, transdermally. In some embodiments, the pharmaceutical compositions are administered orally. Pharmaceutical compositions are administered to the individual for a length of time effective to eliminate, reduce or stabilize viral titer. When used prophylactically, Pharmaceutical compositions are administered to the individual for a length of time during which monitoring for evidence of infection continues.

Pharmaceutical compositions used in the present invention may be administered either as individual therapeutic agents or in combination with other therapeutic agents. They can be administered alone, but are generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical practice.

Dosage varies depending upon known factors such as the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired. Usually a daily dosage of active ingredient can be about 0.001 to 1 grams per kilogram of body weight, in some embodiments about 0.1 to 100 milligrams per kilogram of body weight. Ordinarily dosages are in the range of 0.5 to 50 milligrams per kilogram of body weight, and preferably 1 to 10 milligrams per kilogram per day. In some embodiments, the pharmaceutical compositions are given in divided doses 1 to 6 times a day or in sustained release form is effective to obtain desired results.

Dosage forms (composition) suitable for internal administration generally contain from about 1 milligram to about 500 milligrams of active ingredient per unit. In these pharmaceutical compositions the active ingredient will ordinarily be present in an amount of about 0.5-95 by weight based on the total weight of the composition. Generally, multiple administrations are performed. In some embodiments, dosage forms administered in a 24 hour period is less than 200 milligrams. In some embodiments, dosage forms administered in a 24 hour period is 150 milligrams or less. In some embodiments, dosage forms administered in a 24 hour period is 100 milligrams or less. In some embodiments, dosage forms administered in a 24 hour period is 75 milligrams or less. In some embodiments, dosage forms administered in a 24 hour period is 60 milligrams or less. In some embodiments, dosage forms administered in a 24 hour period is 50 milligrams or less. In some embodiments, dosage forms administered in a 24 hour period is 40 milligrams or less In some embodiments, dosage forms administered in a 24 hour period is 30 milligrams or less. In these pharmaceutical compositions the active ingredient will ordinarily be present in an amount of about 0.5-95 by weight based on the total weight of the composition. Generally, multiple administrations are performed.

According to preferred embodiments of the invention, the compounds are provided over a course of time in which a therapeutically effective amount of compound is present in the individual's body so as to increase platelet levels above suppressed levels, preferably to normal or near normal levels. According to such preferred embodiments, drug titer remains at therapeutically effective levels in the individual for an extended period of time such as 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, 20 days, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 or more days, 48 or more days, 60 or more days or 75 or more days.

Pharmaceutical compositions may be formulated by one having ordinary skill in the art with compositions selected depending upon the chosen mode of administration. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field, which is incorporated herein by reference.

For parenteral administration, the compound can be formulated as a solution, suspension, emulsion or lyophilized powder in association with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils may also be used. The vehicle or lyophilized powder may contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives). The formulation is sterilized by commonly used techniques. In some embodiments, a parenteral composition suitable for administration by injection is prepared by dissolving 1.5% by weight of active ingredient in 0.9% sodium chloride solution.

According to some embodiments of the present invention, the composition is administered to tissue of an individual by topically or by lavage. The compounds may be formulated as a cream, ointment, salve, douche, suppository or solution for topical administration or irrigation. The compounds may be formulated as a transdermal patch or subdermal implants. Formulations for such routes administration of pharmaceutical compositions are well known. Generally, additives for isotonicity can include sodium chloride, dextrose, mannitol, sorbitol and lactose.

In some cases, isotonic solutions such as phosphate buffered saline are used. Stabilizers include gelatin and albumin. In some embodiments, a vasoconstriction agent is added to the formulation. The pharmaceutical preparations according to the present invention are preferably provided sterile and pyrogen free. The pharmaceutical preparations according to the present invention which are to be used as injectables are provided sterile, pyrogen free and particulate free.

A pharmaceutically acceptable formulation will provide the active ingredient(s) in proper physical form together with such excipients, diluents, stabilizers, preservatives and other ingredients as are appropriate to the nature and composition of the dosage form and the properties of the drug ingredient(s) in the foπnulation environment and drug delivery system.

The pharmaceutical compositions according to the present invention may be administered as a single dose or in multiple doses. The pharmaceutical compositions of the present invention may be administered either as individual therapeutic agents or in combination with other therapeutic agents. The treatments of the present invention may be combined with conventional therapies, which may be administered sequentially or simultaneously.

The foregoing embodiments are meant to illustrate the invention and are not to be construed to limit the invention in any way. Those skilled in the art will recognize modifications that are within the spirit and scope of the invention. All references cited herein are hereby incorporated by reference in their entirety.

Claims

1. A method of treating an individual who has been diagnosed as having elevated platelet levels comprising the step of administering to said individual a therapeutically effective amount of an pharmaceutical composition comprising one or more of the components selected from the group consisting of: i) Vpr protein; ii) a function fragment of Vpr protein; iii) a nucleic acid encoding Vpr protein operably linked to regulatory elements; and iv) a nucleic acid encoding a functional fragment of Vpr protein operably linked to regulatory elements.

2. The method of claim 1 wherein the individual is administered a nucleic acid encoding Vpr protein or functional fragment thereof operably linked to regulatory elements.

3. The method of claim 2, wherein the nucleic acid is administered in a dose of 1 to 500 micrograms nucleic acid.

4. The method of claim 2, wherein the nucleic acid is administered in a dose of 25 to 250 micrograms nucleic acid.

5. The method of claim 2, wherein the nucleic acid is administered in a dose of about 100 micrograms nucleic acid.

6. The method of claim 2, wherein the nucleic acid encoding Vpr protein or functional fragment thereof operably linked to regulatory elements is contained in a plasmid.

7. The method of claim 2, wherein the nucleic acid encoding Vpr protein or functional fragment thereof operably linked to regulatory elements is contained in a viral vector.

8. The method of claim 7, wherein the viral vector is selected from the group consisting of a retroviral vector and an adenoviral vector.

9. The method of claim 1, wherein the step of administering the pharmaceutical composition is repeated at least once.

10. The method of claim 9, wherein the step of administering the pharmaceutical composition is undertaken 1 to 6 times a day.

11. The method of claim 1, wherein the step of administering the pharmaceutical composition comprises continuous administration.

12. The method of claim 1 wherein the individual is administered Vpr protein of a functional fragment thereof.

13. The method of claim 11 , wherein the Vpr protein or functional fragment thereof is administered at 0.1 to 100 mg/kg body weight per day.

14. The method of claim 11, wherein the Vpr protein or functional fragment thereof is administered at 0.5 to 50 mg/kg body weight per day.

15. The method of claim 11 , wherein the Vpr protein or functional fragment thereof is administered at 1.0 to 10 mg/kg body weight per day.

16. The method of claim 1 wherein the individual has essential thrombocythaemia.

17. A method of treating an individual who has been diagnosed as having insufficient platelet levels comprising the step of administering to said individual a amount of one or more glucocorticoid receptor antagonist compounds, wherein said compound has steroidal structure effective to increase platelet numbers.

18. The method of claim 17 wherein the individual is infected with HIV.

19. The method of claim 17 wherein said compound has a steroidal structure free of a hydroxy lated group at position 1 lβ.

20. The method of claim 17 wherein mifepristone is administered to said individual.

21. The method of claim 17 wherein the composition administered at least twice.

22. The method of claim 17 wherein the composition administered 1 to 6 times a day.

23. The method of claim 17 wherein the compositions is administered by continuous administration.