Use of thymosin α for treatment of sepsis

Abstract

To provide methods for preventing, treating, or reducing the severity of sepsis, severe sepsis or septic shock, including bacterial, viral and fungal infections and including infections of more complex etiology.SOLUTION: The invention involves the administration of a thymosin α peptide regimen. The thymosin α peptide regimen is scheduled or timed with respect to potential, expected and / or diagnosed sepsis, severe sepsis or septic shock. In a method, the thymosin α peptide is administered at a dose of at least about 0.5 mg, preferably about 1.6 to about 6.4 mg, further preferably about 1.6 to 3.2 mg, per day intravenously or by continuous infusion or subcutaneous injection from 1 to 4 times daily. The patient is immunodeficient or immunocompromised, and the regimen of thymosin α peptide helps to protect the patient from, or reduce the severity of, sepsis, severe sepsis or septic shock.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 61 / 618,563, filed Mar. 30, 2012, U.S. Provisional Patent Application No. 61 / 643,824, filed May 7, 2012, and U.S. Patent Application No. 13 / 835,107, filed Mar. 15, 2013, which are hereby incorporated by reference in their entirety.

[0002] Field of the Invention The present invention relates to the field of sepsis, including prevention of sepsis, reduction of the severity of sepsis, or treatment of sepsis, by administering a regimen of thymosin alpha peptide.

Background Art

[0003] Background Septic shock is a pathological condition in which an infection has spread extensively to many areas of the body. The infection generally spreads through the blood from one tissue to another, causing extensive damage. Septic shock can be caused by a number of conditions, including: (1) peritonitis caused by the spread of infection from the uterus and fallopian tubes; (2) peritonitis resulting from rupture of the intestine, which can be caused by intestinal disease or trauma; (3) septicemia resulting from the spread of a simple infection; (4) a systemic necrotizing infection, particularly from Clostridium perfringens; and (5) an infection that has spread from the kidney, urinary tract, or abdomen into the bloodstream.

[0004] Sepsis frequently occurs as a nosocomial infection, causing significant patient mortality and morbidity, and significantly increasing the total medical cost [Michael Klompas, Prevention of ventilator-associated pneumonia, Expert Rev. Anti Infect. Ther. 8(7), 791-800 (2010)(Non-Patent Document 1); Wheeler DS et al., Novel Pharmacologic Approaches to the Management of Sepsis: Targeting the Host Inflammatory Responses, Recent Pat. Inflamm. Allergy Drug Discov. 3(2):96-112 (2009)(Non-Patent Document 2)]. In fact, sepsis was reported as the 10th leading cause of death in 2004 (see, for example, Non-Patent Document 2). In fact, in the United States, 750,000 people are diagnosed with severe sepsis annually, and 215,000 of them die due to severe sepsis (see Angus DC, et al., Epidemiology of severe sepsis in the United States: analysis of incidence, outcome, and associated costs of care. Crit Care Med., 29:1303-1310 (2001)(Non-Patent Document 3)).

[0005] A strong and rapid immune response against pathogens is important for preventing, treating, and / or reducing the severity of sepsis caused by viral, bacterial, and fungal infections. Means that can reduce the impact of infection and help prevent, reduce, or treat sepsis are highly sought after.

Prior Art Documents

Non-Patent Documents

[0006]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Summary of the Invention

[0007] The present invention provides a method for treating sepsis. The present invention includes the administration of a thymosin α peptide regimen, and the administration provides a statistically significant therapeutic effect for the treatment of sepsis.

[0008] In some embodiments, the subject is human. In some embodiments, the subject is immunocompromised.

[0009] In some embodiments, the sepsis is due to a nosocomial infection. In some embodiments, the sepsis is due to a bacterial, fungal, or viral infection.

[0010] In some embodiments, the subject exhibits one or more signs or symptoms of an infectious disease. In some embodiments, the subject exhibits one or more signs or symptoms of sepsis.

[0011] In some embodiments, the thymosin α peptide is administered at least about 0.5 mg per day, or about 0.5 to about 3 mg per day, or about 1.6 to about 3.2 mg per day, or at least about 1.6 mg per day.

[0012] In some embodiments, the thymosin α peptide is administered intravenously. In some embodiments, the thymosin α peptide is administered by continuous infusion or subcutaneous injection.

[0013] In some embodiments, the thymosin α peptide is administered about 1 to 4 times per day. In some embodiments, the thymosin α peptide is administered approximately 2 times per day. In some embodiments, the thymosin α peptide is administered approximately 1 time per day. In some embodiments, the thymosin α peptide is administered about 2 times per day for at least 5 days (e.g., 5 to 14 days). In some embodiments, the thymosin α peptide is administered about 2 times per day for about 5 to 10 days (or about 5 days), then about 1 time per day for at least 2 days, or about 2 to 7 days, or about 2 days.

[0014] In another aspect, the present invention provides a method for treating sepsis by administering a regimen of thymosin α peptide. In this aspect, the patient has been diagnosed as suffering from sepsis. The sepsis can be due to bacterial, viral, fungal or mixed or unknown etiology.

[0015] In some embodiments, sepsis is associated with infectious microorganisms selected from Lysteria monocytogenes, Pseudomonas sp. (e.g., P. aeruginosa), Serratia marcescens, Clostridium difficile, Staphylococcus aureus, Staphylococcus sp., Acinetobacter spp., Enterococcus sp., Enterobacter sp., E. coli, Klebsiella sp., Streptococcus (e.g., S. pneumoniae), Haemophilus influenzae, and Neisseria meningitidis.

[0016] In some embodiments, sepsis is associated with one or more drug-resistant microorganisms, such as Staphylococcus aureus, Staphylococcus sp., Enterococcus sp., Pseudomonas sp., Klebsiella sp., E. coli, and / or Clostridium difficile. In some embodiments, sepsis is associated with methicillin-resistant or vancomycin-resistant Staphylococcus aureus, including moderately resistant isolates, and / or carbapenem-resistant E. coli, Klebsiella, or Pseudomonas, including moderately resistant isolates.

[0017] The thymosin alpha peptide regimen can be administered concurrently with standard treatments, such as antibiotic therapy or antiviral therapy. According to this aspect of the invention, the thymosin alpha peptide regimen reduces the duration of sepsis and / or the duration of antibacterial, antiviral, or antifungal treatment required.

[0018] [Invention 1001] A method for treating sepsis in a subject, comprising the step of administering a regimen of thymosin alpha peptide, wherein the administration provides a statistically significant therapeutic effect for the treatment of sepsis. [Inventive concept 1002] The method of Inventive concept 1001, wherein the subject is a human. [Inventive concept 1003] The method of Inventive concept 1001, wherein the subject is immunocompromised. [Inventive concept 1004] The method of Inventive concept 1001, wherein the sepsis is caused by a nosocomial infection. [Inventive concept 1005] The method of Inventive concept 1001, wherein the sepsis is caused by a bacterial, fungal or viral infection. [Inventive concept 1006] The method of Inventive concept 1001, wherein the thymosin alpha peptide is administered at a dose of at least about 0.5 mg per day. [Inventive concept 1007] The method of Inventive concept 1001, wherein the thymosin alpha peptide is administered at a dose of about 1.6 to about 6.4 mg per day. [Inventive concept 1008] The method of Inventive concept 1001, wherein the thymosin alpha peptide is administered at a dose of at least about 1.6 mg or 3.2 mg per day. [Inventive concept 1009] The method of Inventive concept 1001, wherein the thymosin alpha peptide is administered intravenously. [Inventive concept 1010] The method of Inventive concept 1001, wherein the thymosin alpha peptide is administered by continuous infusion. [Inventive concept 1011] The method of Inventive concept 1001, wherein the thymosin alpha peptide is administered by subcutaneous injection. [Inventive concept 1012] The method of Inventive concept 1001, wherein the regimen comprises administering the thymosin alpha peptide 1 to 4 times per day. [Inventive concept 1013] The method of Inventive concept 1001, wherein the thymosin alpha is administered approximately 2 times per day. [Inventive concept 1014] The method of Inventive concept 1001, wherein the thymosin alpha peptide is administered approximately 1 time per day. [The present invention 1015] The method of the present invention 1001, wherein the thymosin α peptide is administered twice a day for at least 5 days. [The present invention 1016] The method of the present invention 1001, wherein the thymosin α peptide is administered twice a day for about 5 to 14 days. [The present invention 1017] The method of the present invention 1001, wherein the thymosin α peptide is administered twice a day for at least 5 days and then once a day for at least 2 days. [The present invention 1018] The method of the present invention 1001, wherein the thymosin α peptide is administered twice a day for about 5 to 14 days and then once a day for about 2 to 7 days. [The present invention 1019] The method of the present invention 1001, wherein the subject exhibits one or more signs or symptoms of infection. [The present invention 1020] The method of the present invention 1001, wherein the subject exhibits one or more signs or symptoms of sepsis. [The present invention 1021] The method of the present invention 1001, wherein the thymosin α peptide is administered within at least the first 24 hours, 48 hours, 72 hours, or 96 hours after the subject exhibits one or more signs or symptoms of infection or sepsis. [The present invention 1022] The method of the present invention 1001, wherein sepsis is confirmed by a diagnostic test. [The present invention 1023] The method of the present invention 1001, wherein the regimen of the thymosin α peptide is administered concurrently with antibacterial therapy, antiviral therapy, or antifungal therapy. [The present invention 1024] The method of the present invention 1001, wherein sepsis is associated with an infectious microorganism selected from the group consisting of Lysteria monocytogenes, Pseudomonas sp. (e.g., Pseudomonas aeruginosa), Serratia marcescens, Clostridium difficile, Staphylococcus aureus, Staphylococcus sp., Acinetobacter spp., Enterococcus sp., Enterobacter sp., Escherichia coli, Klebsiella sp., Streptococcus (e.g., Streptococcus pneumoniae), Haemophilus influenzae, and Neisseria meningitidis. [The present invention 1025] The method of the present invention 1001, wherein sepsis is associated with a drug-resistant or multi-drug resistant Staphylococcus aureus, Staphylococcus sp., Enterococcus sp., Pseudomonas sp., Klebsiella sp., Escherichia coli, or Clostridium difficile. [The present invention 1026] The method of the present invention 1001, wherein sepsis is associated with methicillin-resistant or vancomycin-resistant Staphylococcus aureus. Other objects and aspects of the present invention will become apparent from the following detailed description.

Brief Description of the Drawings

[0019]

Figure 1

Figure 2

Figure 3

Mode for Carrying Out the Invention

[0020] Detailed Description of the Invention The present invention is based in part on the discovery that sepsis can be treated using a specific regimen of thymosin α peptide therapy, in particular that a thymosin α peptide therapy administered at a specific low dose can provide a statistically significant therapeutic effect for the treatment of sepsis. Accordingly, the present invention provides a method for treating sepsis by administering a regimen of thymosin α peptide to a subject, wherein the administration provides a statistically significant therapeutic effect for the treatment of sepsis.

[0021] According to the present invention, sepsis includes any recognized form of sepsis, such as sepsis due to nosocomial infection, sepsis related to medical procedures, sepsis related to medical devices, severe sepsis, or septic shock. Sepsis also includes any recognized condition or symptom related to sepsis. Generally, the symptoms of sepsis include, but are not limited to, fever above 101.3°F (38.5°C) or below 95°F (35°C), heart rate over 90 beats per minute, respiratory rate over 20 breaths per minute, and a likely or confirmed infection (i.e., the presence of one or more infectious pathogens such as bacteria, fungi, or viruses). Typically, a clinical diagnosis of sepsis includes the presence of at least two symptoms selected from the symptoms of sepsis. The symptoms of severe sepsis include, but are not limited to, significantly decreased urine output, a rapid change in mental status, a decrease in platelet count, difficulty breathing, abnormal heart pump function, and abdominal pain. Typically, a clinical diagnosis of severe sepsis includes the presence of at least one additional symptom selected from the symptoms of severe sepsis, the presence of which indicates organ failure. The symptoms of septic shock can include, but are not limited to, extremely low blood pressure that does not respond to simple fluid resuscitation. Typically, a clinical diagnosis of septic shock includes the presence of at least one additional symptom selected from the symptoms of septic shock.

[0022] Generally, sepsis can be caused by a variety of infectious pathogens including bacteria, fungi, viruses, and parasites, and if untreated, can progress from a simple infection to multiple organ dysfunction syndrome (MODS) and ultimately death. In some embodiments, sepsis can include bacteremia or fungal infections such as candidemia or aspergillus infections. In some embodiments, sepsis can be caused by severe injury, severe trauma, or burns and can be a postoperative infection.

[0023] According to the present invention, the treatment of sepsis includes any form of treatment or prevention of sepsis, for example, reducing any symptom of sepsis, reducing the severity of any symptom of sepsis, delaying the onset of sepsis, shortening the duration of one or more symptoms of sepsis, reducing the chance or occurrence of sepsis, treating or inhibiting any cause or condition associated with sepsis, reducing any clinical criterion or measurement of the degree or condition of sepsis (e.g., ICU frequency, length of stay in the ICU, number of days not requiring ICU, duration of mechanical ventilation, number of days not requiring mechanical ventilation, mortality rate (e.g., 28-day mortality, in-ICU mortality, in-hospital mortality, etc.), dynamic changes in SOFA, HLA-DR expression, etc.).

[0024] In one aspect, the present invention includes administering a regimen of thymosin α peptide to enhance the immune response to exposure to a pathogen or potential pathogen in order to treat sepsis.

[0025] Thymosin α was originally isolated from bovine thymus and has been shown to "reconstitute immune function" in thymectomized animal models. Thymosin is thought to play a role in the inflammatory and innate immune responses and to facilitate the discrimination between non-self and self in mammals. Activation of specific Toll-like receptors (TLRs; also known as pathogen-associated molecular patterns or PAMPs) by thymosin stimulates intracellular signaling pathways, resulting in the expression of co-stimulatory molecules, pro-inflammatory cytokines, nitric oxide, and eicosanoids. Thymosin can affect, for example, progenitor cells, dendritic cells, T cells, B cells, and NK cells.

[0026] Although not intended to be bound by theory, thymosin alpha peptide (e.g., TA1) is thought to activate Toll-like receptor 9 (TLR), resulting in an increase in Th1 cells, B cells, and NK cells, thereby stimulating the immune system for enhanced immune response. For example, TA1 can increase or enhance lymphocyte infiltration, chemotactic cytokine secretion, dendritic cell maturation and differentiation, secretion of thymopoeitic cytokines including IFN-α, IL-7, and IL-5, and antibody production by B cells.

[0027] According to the present invention, the thymosin alpha peptide used in the method of the present invention includes thymosin alpha 1 ("TA1"; "Tα1"), and peptides having structural homology to TA1. TA1 has the amino acid sequence TIFF2025107346000001.tif13144. The amino acid sequence of TA1 is disclosed in U.S. Patent No. 4,079,127, the disclosure of which is incorporated herein by reference. TA1 is an unglycosylated 28-amino acid peptide with an acetylated N-terminus and a molecular weight of about 3108. Synthetic form of TA1 is commercially available under the trade name ZADAXIN in certain countries.

[0028] In some embodiments, thymosin alpha peptides suitable for the methods of the invention include natural TA1 (e.g., TA1 purified or isolated from tissue), synthetic TA1, recombinant TA1, and any suitable TA1 analogs having substantially the same or better function as TA1. In some other embodiments, the thymosin peptide comprises the amino acid sequence of SEQ ID NO: 1 (optionally with an acylated, e.g., acetylated, N-terminus). In some embodiments, the thymosin peptide comprises an amino acid sequence substantially similar to TA1 and maintains the immunomodulatory activity of TA1. A substantially similar sequence may have, for example, from about 1 to about 10 amino acid deletions, insertions, and / or substitutions (collectively) relative to TA1. For example, the thymosin peptide may have from about 1 to about 5 (e.g., 1, 2, or 3) amino acid insertions, deletions, and / or substitutions (collectively) relative to TA1.

[0029] In some embodiments, the thymosin α peptide may comprise a truncated TA1 sequence having a deletion of from about 1 to about 10 amino acids, or from about 1 to 5 amino acids, or 1, 2, or 3 amino acids, for example, relative to TA1. Such deletions may be at the N-terminus and / or C-terminus and / or internally, so long as the immunomodulatory activity of the peptide is substantially maintained. Alternatively, or in addition, a substantially similar sequence may have an insertion of from about 1 to about 5 amino acids (e.g., an insertion of 1, 2, or 3 amino acids) relative to TA1, if the immunomodulatory activity of TA1 is substantially maintained. Alternatively, or in addition, a substantially similar sequence may have a substitution of from 1 to about 10 amino acids if the immunomodulatory activity is substantially maintained. For example, a substantially similar sequence may have a substitution of from 1 to about 5, or 1, 2, or 3 amino acids, which may include conservative and non-conservative substitutions. In some embodiments, the substitutions are conservative. Generally, conservative substitutions include the substitution of chemically similar (e.g., polar, non-polar, or charged) amino acids. The substituted amino acids may be selected from the standard 20 amino acids or may be non-standard amino acids (e.g., conserved non-standard amino acids).

[0030] In some embodiments, the thymosin α peptide comprises a TA1 sequence substituted with one or more non-natural or modified amino acids. In some other embodiments, the thymosin α peptide comprises a TA1 sequence conjugated to one or more entities. In some embodiments, the thymosin α peptide is pegylated to increase its half-life in circulation. Such strategies for increasing the half-life of therapeutic proteins are well known.

[0031] In some embodiments, thymosin peptide comprises an amino acid sequence having at least 70% sequence identity to SEQ ID NO:1 while maintaining the immunomodulatory activity of TA1. For example, the thymosin peptide may comprise an amino acid sequence having at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% or 97% sequence identity to SEQ ID NO: 1. The thymosin peptide may comprise an amino acid sequence having 100% sequence identity to SEQ ID NO: 1. In all cases, the N-terminus may be optionally acylated (e.g., acetylated) or alkylated with, for example, a C1-C10 or C1-C7 acyl or alkyl group.

[0032] In certain embodiments, the above-described substantially similar and homologous peptides can function at levels of at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96% or 97% compared to TA1 (SEQ ID NO: 1).

[0033] Generally, thymosin alpha peptide may be prepared synthetically, e.g., by solid-phase synthesis, or may be made recombinantly by known techniques and purified.

[0034] In some embodiments, the thymosin alpha peptide may be provided in lyophilized form and reconstituted with a sterilized (e.g., aqueous) diluent prior to administration.

[0035] According to the present invention, the thymosin alpha peptide of the present invention is administered in a regimen for the treatment of sepsis. Such regimens include the dose per administration, the dose per day, and the number of days per treatment cycle, or combinations thereof.

[0036] Generally, thymosin α can be administered at a dosage of about 0.2 mg to 20 mg, 0.2 mg to 15 mg, 0.4 to 10 mg, 0.5 mg to 8 mg, 0.5 mg to 6 mg, 0.5 mg to 3 mg. In some embodiments, thymosin α is administered at 0.2 mg, 0.5 mg, 0.4 mg, 0.8 mg, 1 mg, 1.6 mg, 3 mg, 3.2 mg, 6.4 mg or about 8 mg. In some embodiments, the thymosin α peptide is administered to a human patient at a dosage corresponding to at least about 0.5 mg (e.g., at least about 0.8 mg, or at least about 1.6 mg), at least about 3 mg (e.g., at least about 3.2 mg) or at least about 5 mg (e.g., at least about 6.4 mg) of TA1. In some embodiments, the thymosin peptide is administered within a range corresponding to about 0.1 to 20 mg of TA1, or about 1 to 10 mg of TA1, or about 2 to 10 mg of TA1, or about 2 to 8 mg of TA1, or about 2 to 7 mg of TA1. In certain embodiments, the dosage unit is within the range of about 3 to 6.5 mg, e.g., about 3.2 or 6.4 mg of TA1. In certain embodiments, the dosage of TA1 is adjusted according to the size of the patient and may be provided at 10 to 100 μg / kg (e.g., about 20, 40, 60, or 80 μg / kg). The dosage may also be adjusted for each patient's condition and other drugs being taken by the patient. In addition, the dosage may be adjusted according to the species of the subject, but in each case, it approximately corresponds to the human equivalent amount (mg / kg) of TA1.

[0037] In some embodiments, such dosages are administered hourly, daily, weekly or monthly.

[0038] In some embodiments, the thymosin α peptide is administered hourly, every about 1 to 24 hours, every 1 to 20 hours, every 1 to 16 hours, every 1 to 12 hours, every 1 to 8 hours, every 1 to 6 hours, every 1 to 4 hours, every 1 to 2 hours, or every 1 hour. In some embodiments, the thymosin α peptide is administered every about 2, 3, 5, 5 or 6 hours, or every about 10 minutes, 15 minutes, 30 minutes, 45 minutes, or 60 minutes.

[0039] Alternatively, thymosin peptide can be administered by multiple injections (split doses of thymosin peptide) on the treatment day so that an immunostimulatory effective amount of thymosin peptide is maintained substantially continuously in the patient's circulatory system for a longer period of time. Suitable injection regimens may include injections (e.g., 2 to 5 injections) every 2, 3, 4, 6 hours, etc. on the day of administration so that an immunostimulatory effective amount of thymosin peptide is maintained substantially continuously in the patient's circulatory system on the day of treatment with thymosin.

[0040] In some embodiments, TA1 may be administered by continuous infusion. Continuous infusion of TA1 is described in detail in US2005 / 0049191, the entire disclosure of which is incorporated herein by reference. Briefly, continuous infusion of thymosin peptide maintains an immunostimulatory effective amount of thymosin peptide in the patient's circulatory system for a longer period of time. In some embodiments, thymosin peptide may be administered to the patient over a treatment period of at least about 2, 4, 6, 10, 12 hours or more, which may improve efficacy in some embodiments. The infusion may be carried out by any suitable means such as by a minipump.

[0041] In some embodiments, thymosin α is administered by continuous infusion over a period of about 1 to 168 hours, 1 to 144 hours, 1 to 120 hours, 1 to 96 hours, 1 to 72 hours, 1 to 48 hours, 1 to 24 hours, 1 to 20 hours, 1 to 16 hours, 1 to 12 hours, 1 to 10 hours, 1 to 8 hours, 1 to 6 hours, 1 to 4 hours to 1 to 2 hours. In some embodiments, the thymosin α peptide is administered by continuous infusion over a period of about 10 minutes, 15 minutes, 30 minutes, 45 minutes, or 60 minutes. In some embodiments, thymosin α is administered by continuous infusion over a period of about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 10 hours, 12 hours, 24 hours or more. In some embodiments, the continuous infusion period is interrupted by a non-infusion period (i.e., a period during which no thymosin α is administered). In some embodiments, the non-infusion period ranges from 1 to 168 hours, 1 to 144 hours, 1 to 120 hours, 1 to 96 hours, 1 to 72 hours, 1 to 48 hours, 1 to 24 hours, 1 to 20 hours, 1 to 16 hours, 1 to 12 hours, 1 to 10 hours, 1 to 8 hours, 1 to 6 hours, 1 to 5 hours, 1 to 4 hours, 1 to 3 hours, 1 to 2 hours. In some embodiments, the non-infusion period is about 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 10 hours, 12 hours, 24 hours or more.

[0042] In some embodiments, a predetermined amount of thymosin α peptide, e.g., an immunostimulatory effective amount of thymosin peptide (e.g., TA1), may be substantially continuously maintained in the patient's circulatory system by administering the TA1 peptide to the patient at a rate within the range of about 0.0001 to 0.1 mg / hr / Kg patient body weight. Exemplary infusion rates are within the range of about 0.0003 to 0.03 mg / hr / Kg patient body weight. For continuous infusion, the TA1 peptide is present in a pharmaceutically acceptable liquid carrier, e.g., water for injection or physiological concentration of saline.

[0043] In some embodiments, thymosin is administered about every 1 to 20 days, every 1 to 15 days, every 1 to 10 days, every 1 to 7 days, every 1 to 5 days, every 1 to 3 days, or daily. In some embodiments, thymosin α is administered for about 1 to 100 days, 1 to 90 days, 1 to 80 days, 1 to 70 days, 1 to 50 days, 1 to 40 days, 1 to 30 days, 1 to 20 days, 1 to 15 days, 1 to 10 days, 1 to 7 days, 1 to 5 days, 1 to 3 days, 1 to 14 days, 5 to 14 days, or 1 to 2 days. In some embodiments, thymosin α is administered for about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 days or more. In some embodiments, the thymosin α peptide is administered about twice a day for at least 5 days (e.g., 5 to 14 days). In some embodiments, the thymosin α peptide is administered about twice a day for about 5 to 10 days (or about 5 days), and then about once a day for at least 2 days, or about 2 to 7 days, or about 2 days.

[0044] In some embodiments, thymosin α is administered for about 1 to 8 weeks, about 1 to 6 weeks, about 1 to 5 weeks, about 1 to 4 weeks, about 2 to 4 weeks, or about 1 to 2 weeks. In some embodiments, thymosin is administered for 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks or more. In some embodiments, thymosin is administered for about 1 month, 2 months, 3 months or 4 months or more. In some embodiments, the thymosin α peptide is administered for about 1 to 4 months, 1 to 3 months, 1 to 2 months, or about 1 month.

[0045] In some embodiments, the thymosin α peptide is administered about 1 to 8 times a day for about 1 to 8 weeks. In some embodiments, the thymosin α peptide is administered about 1 to 7 times a day, 1 to 6 times a day, 1 to 5 times a day, 1 to 4 times a day, 1 to 3 times a day, 1 to 2 times a day, or about 1 time a day for about 1 to 7 weeks, 1 to 6 weeks, 1 to 5 weeks, 1 to 4 weeks, 1 to 3 weeks, 1 to 2 weeks, or about 1 week. In some embodiments, the thymosin α peptide is administered about 1 to 8 times a day, 1 to 7 times a day, 1 to 6 times a day, 1 to 5 times a day, 1 to 4 times a day, 1 to 3 times a day, 1 to 2 times a day, or about 1 time a day for about 1 to 30 days, 1 to 25 days, 1 to 20 days, 1 to 15 days, 1 to 7 days, or 1 to 5 days. In some embodiments, the thymosin α peptide is administered 1 to 4 times a day for 1 to 30 days. In some embodiments, the thymosin α peptide is administered about 1 to 2 times a day for 1 to 15 days, or 1 to 7 days, or 1 to 5 days. In some embodiments, the thymosin α peptide is administered about 1 to 2 times a day for 5 days, then 1 time a day for 2 days. In some embodiments, the thymosin α peptide is administered about 2 times a day for 5 days, then 1 time a day for 2 days. In some embodiments, thymosin α is administered about 4 times a day for 5 or 7 days.

[0046] In some embodiments, the regimen uses one dose of thymosin alpha peptide that is at least 0.2 mg, 0.5 mg, 0.8 mg, 1.6 mg, 3.2 mg, or 6.4 mg, one, two, three, four, five, six, seven, or eight times or more. In some embodiments, three or fewer doses can be administered. In some embodiments, more doses, for example, five, six, seven, eight, nine or ten, eleven, twelve, thirteen, fourteen, fifteen, twenty, twenty-five, thirty, thirty-five, forty, forty-five, fifty or more doses may be administered. In some embodiments, the dose of thymosin is a relatively low dose of at least 0.2 mg, 0.4 mg, 0.5 mg, 0.8 mg or 1.6 mg. Administration of thymosin alpha may be spaced about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 20 or 24 hours or about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, as described in more detail herein, and in some embodiments, may be administered once a week. In some embodiments, the thymosin peptide (e.g., TA1) is administered at a dose in the range of about 0.5 mg to 3 mg. In some embodiments, the thymosin peptide (e.g., TA1) is administered at a dose in the range of about 1 mg to 2 mg.

[0047] In some embodiments, thymosin peptide is administered at a dose of about 0.5 mg, about 0.8 mg, about 1.6 mg, about 3 mg, about 3.2 mg, about 5 mg, or about 6.4 mg or more of thymosin peptide, and optionally in combination with one or more treatment schedules described in this paragraph. In some embodiments, thymosin alpha peptide is administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times, eleven times, twelve times, thirteen times, fourteen times, fifteen times, sixteen times, seventeen times, eighteen times, nineteen times, twenty times, twenty-five times or thirty times a day, or more, for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25 or 30 days or more. In some embodiments, thymosin alpha peptide is administered for 1, 2, 3, 4, 5, 6, 7 or 8 weeks or more. In some embodiments, thymosin is administered for 1 or 2 months or more. In some embodiments, thymosin peptide is administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times, eleven times, twelve times, thirteen times, fourteen times, fifteen times, sixteen times, seventeen times, eighteen times, nineteen times, twenty times, twenty-five times or thirty times a day, or more, for 2, 3, 4, 5, 6, 7 or 8 days. In some embodiments, thymosin peptide is administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times, eleven times, twelve times, thirteen times, fourteen times, fifteen times, sixteen times, seventeen times, eighteen times, nineteen times, twenty times, twenty-five times or thirty times a day, or more, for 4, 5, 6 or 7 days. In some embodiments, thymosin peptide is administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times, eleven times, twelve times, thirteen times, fourteen times, fifteen times, sixteen times, seventeen times, eighteen times, nineteen times, twenty times, twenty-five times or thirty times a day, or more, for 5, 6, or 7 days. In some embodiments, thymosin peptide is administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times, eleven times, twelve times, thirteen times, fourteen times, fifteen times, sixteen times, seventeen times, eighteen times, nineteen times, twenty times, twenty-five times or thirty times a day, or more, for 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days or more, and then once, twice, three times, or four times, or more, for 1, 2, 3, or 4 days or more. In some embodiments, thymosin peptide is administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times, eleven times, twelve times, thirteen times, fourteen times, fifteen times, sixteen times, seventeen times, eighteen times, nineteen times, twenty times, twenty-five times or thirty times a day, or more, for 2, 3, 4, 5, 6, 7, or 8 days, and then once, twice, three times, or four times, or more, for 1, 2, 3, or 4 days. In some embodiments, thymosin peptide is administered once, twice, three times, four times, five times, six times, seven times, eight times, nine times, or ten times, eleven times, twelve times, thirteen times, fourteen times, fifteen times, sixteen times, seventeen times, eighteen times, nineteen times, twenty times, twenty-five times or thirty times a day, or more, for 4, 5, 6 or 7 days, and then once, twice, three times, or four times, or more, for 1, 2, 3, or 4 days. In some embodiments, thymosin peptide is administered once, twice, or three times a day for 4, 5, 6 or 7 days, followed by once, twice, three times, or four times a day for 1, 2, 3, or 4 days.In some embodiments, thymosin peptide is administered twice a day for 7 days. In some embodiments, thymosin peptide is administered twice a day for 5 days. In some embodiments, thymosin peptide is administered once a day for 5 days. In some embodiments, thymosin peptide is administered twice a day for 5 days and then once a day for 2 days. In some embodiments, about 1.6 mg of thymosin peptide is administered twice a day for 5 days and then once a day for 2 days.

[0048] The timing of thymosin administration may be selected to enhance the immune response, including antibody titers, to include periods of high risk of sepsis (e.g., development of the level of antibody titers). For example, in certain embodiments, thymosin peptide is administered at intervals of about 5 to about 9 days, and in various embodiments, at intervals of about 1, 2, 3, 4, 5, 6, 7, or 8 days. Thymosin may be administered at intervals of about 7 days (e.g., once a week). In other embodiments, thymosin peptide is administered at intervals of 1, 2, 3, or 4 days.

[0049] In other embodiments, the regimen can be initiated from about 1 to 10 days (in some embodiments 5 to 9 days) prior to an event predicted to result in (or having a significant risk of) sepsis, to provide treatment / prevention of sepsis. Exemplary events are described herein. In some of these embodiments, an effective regimen includes about 1 to 5 administrations, such as 3 or fewer administrations, of thymosin alpha peptide. The administrations of thymosin alpha peptide may be at intervals of about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, and in some embodiments may be performed once a week.

[0050] In some embodiments, the thymosin α peptide is first administered before an event (such as described) such as hospitalization in a medical institution, a scheduled surgery, or placement of an invasive medical device, and is administered again on the day of the event and optionally after the event. For example, the thymosin peptide may be administered 1 to 10 days before the event, such as about 5 to about 9 days before the event, and may be administered again on the day of the event. The thymosin peptide may be administered about 7 days before the event, again on the day of the event, and optionally within 2 to 10 days after the event (e.g., 4 to 8 days after the event). For example, a patient receiving two doses of TA1 according to certain embodiments of the invention may achieve a more rapid and / or greater response to sepsis, which may be protective for at least 21 days, at least 42 days, or longer.

[0051] In some embodiments, the thymosin α peptide is administered before, with, and / or after an event predicted to result in exposure to a pathogen or introduction of an opportunistic environment, as described herein. For example, the event can be hospitalization in a hospital or medical institution for a period of time (e.g., at least 3 days, at least 1 week, or at least 10 days, or at least 1 month). In other embodiments, the event is a scheduled surgery or invasive medical procedure as described. In other embodiments, the event is placement of an invasive medical device as described. In yet other embodiments, the event is the initiation of (as described) renal dialysis or chemotherapy or radiation therapy for cancer treatment.

[0052] In some embodiments, thymosin is administered within about 1 hour, 2 hours, 4 hours, 6 hours, 8 hours, 10 hours, 12 hours, 24 hours, 72 hours, 96 hours, 120 hours, 144 hours, or 168 hours after determination of sepsis. In some embodiments, the thymosin α peptide is administered within about 10 minutes, 15 minutes, 30 minutes, 45 minutes, or 60 minutes after determination of sepsis.

[0053] In yet other embodiments, the regimen comprises administration of thymosin alpha peptide one to four times, such as three times or less, and the regimen is timed to begin before an event that is predicted to lead to sepsis. For example, the regimen may be initiated 2 to 10 days, such as 5 to 10 days, before the event, and the second dose may be administered on the day of the event. Administration of thymosin alpha peptide may be at intervals of about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, and in some embodiments may be performed once a week. In yet other embodiments, the regimen comprises one dose of thymosin alpha peptide provided approximately once a week (e.g., every 5 to 9 days) over 2, 3, 4 weeks or more.

[0054] In yet other embodiments, the patient receives two doses of thymosin alpha peptide (e.g., 2 mg to 8 mg per dose), and such doses are at intervals of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 20 or 24 hours or about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 days, or approximately once a week. This regimen may be repeated approximately once a month or every other month, and may be particularly beneficial for protecting patients with chronic diseases and immunodeficiencies from sepsis. In some embodiments, the thymosin peptide (e.g., TA1) is administered at a dose in the range of about 0.5 mg to 3 mg. In some embodiments, the thymosin peptide (e.g., TA1) is administered at a dose in the range of about 1 mg to 2 mg.

[0055] In certain aspects of the invention, the thymosin alpha peptide regimen is part of an in - facility program to reduce the rate or incidence of sepsis, such as nosocomial sepsis.

[0056] In some embodiments, the thymosin alpha peptide regimen comprises administering the agent to the subject at a dose sufficient to raise antibody titers and / or accelerate the rise in antibody titers in response to exposure to a pathogen. In some other embodiments, the thymosin alpha peptide regimen comprises a regimen that provides a serum level of thymosin alpha of about 0.01-10.0 ng / ml, 0.1-1.0 ng / ml, or 0.05-5 ng / ml during treatment. In some embodiments, the peak plasma level of thymosin alpha peptide is at least about 10 ng / ml, 20 ng / ml, 30 ng / ml, 40 ng / ml, 50 ng / ml, 60 ng / ml, 70 ng / ml, 80 ng / ml, 90 ng / ml, or 100 ng / ml. In some other embodiments, the thymosin alpha peptide regimen includes administering the agent to the subject in a regimen such that the subject's pharmacokinetic (pK) profile is substantially the same, e.g., within at least 60%, 70%, 80%, 90%, of the pK profile of a subject treated with 1.6 mg thymosin alpha twice daily for 5 days followed by once daily for 2 days. In another embodiment, the pK profile is increased to greater than 100% of the pK profile of a subject treated with 1.6 mg thymosin alpha twice daily for 5 days followed by once daily for 2 days.

[0057] The thymosin peptide may be provided in lyophilized form and may be reconstituted with a sterile (e.g., aqueous) diluent prior to administration. The thymosin peptide (e.g., TA1) may be administered by any effective route, including subcutaneous injection, intramuscular injection, intravenous injection or infusion, and orally. In certain embodiments, the thymosin peptide is administered by subcutaneous injection or intravenous infusion. In general, the planned dose of thymosin may be administered (e.g., injected) as a single dose, or may be spaced apart over a period of 24 hours or less, such as by continuous infusion or repeated injections of divided doses, or as fully described herein. In one embodiment, the planned dose of thymosin peptide may be administered as a single injection or as multiple injections.

[0058] In some embodiments, the thymosin α peptide is administered twice daily for a period of time at one dose and then once daily for a period of time at the same dose. For example, according to the present invention, the thymosin α peptide is administered twice daily at about 1.6 mg for 5 days and then once daily at 1.6 mg for 2 days. In some embodiments, the thymosin α peptide is administered 3 or 4 times daily at about 1.6 mg for 5 to 7 days and then once or twice daily for 2 or 4 days.

[0059] In certain embodiments, a patient receives TA1 at a dose of 2 - 8 mg (e.g., 0.8, 1.6, 3.2, or 6.4 mg per dose) once or twice daily or every other day for 3 - 14 days (e.g., 3, 5, 7, 10, or 14 days). Such regimens may be timed relative to events that expose the patient to an increased risk of exacerbating an infection or complication, such as those described herein (e.g., surgery, hemodialysis, initiation of cancer treatment, placement of a medical device). For example, the event may be scheduled at a time between day 2 and day 10, including day 3, day 5, day 7, or day 10 of the regimen. The regimen may be used in combination with antibacterial, antiviral, or antifungal therapy, including the active agents described herein. In some embodiments, thymosin is administered within the first 24, 48, 72, 96, 120, or 144 hours.

[0060] In one embodiment, a patient receives administration of TA1 at a dose of 0.5 - 8 mg (e.g., about 0.8, 1.6, 3.2, or 6.4 mg) once approximately every week to protect against sepsis or reduce the severity of sepsis. The regimen may be continued for 2 - 4 weeks in some embodiments. When a patient participates in a TA1 program at a medical institution, the present invention provides a reduction in the occurrence of sepsis, a reduction in the length of stay in the ICU, and / or a reduction in antimicrobial therapy.

[0061] In accordance with the present invention, the thymosin α peptide of the present invention is administered to a subject using a regimen sufficient to treat sepsis. The thymosin α peptide regimens in some embodiments are "effective" regimens. That is, the regimen achieves its goal by administering the thymosin α peptide relatively infrequently and / or by adjusting the timing of administration of the thymosin α peptide in accordance with events that are predicted to result in sepsis. An "event" is not vaccination, but rather exposure to a potential infectious pathogen or an increased susceptibility to an infectious pathogen that may or does in fact result in sepsis, severe sepsis, or septic shock. An effective regimen of thymosin α peptide is not only relatively simple and comfortable for the patient, but also more affordable and effective.

[0062] According to the present invention, the thymosin α peptide used in the method of the present invention can be administered alone, in combination with standard treatment for sepsis, or as part of a treatment regimen that includes standard treatment for sepsis. In some embodiments, the standard treatment is a protease inhibitor, activated protein C, corticosteroids, a synthetic colloid (pentastarch) for intensive insulin therapy, drotrecogin alfa (activated; DrotAA), volume resuscitation, hydrocortisone, and fludrocortisone. (See, for example, Hotchkiss, R. S. and Karl, I. E., The Pathophysiology and treatment of Sepsis, NEJM, 348:2 (2008)).

[0063] The method provided by the present invention is applicable to the health of both humans and animals. Thus, the subject is generally a mammal, such as a human, a domestic animal (e.g., cow, horse, pig, sheep, etc.), or a pet mammal (e.g., cat or dog). The terms "subject" and "patient" and their derivatives can be used interchangeably for the method of the present invention.

[0064] In certain embodiments, the subject is immunocompromised. Immunocompromised subjects (e.g., human subjects) exhibit a reduced ability to fight infections and / or a reduced ability to respond to pathogen exposure. Examples of such immunocompromised subjects include elderly patients, neonates, leukemia or neutropenic patients, patients undergoing hemodialysis (e.g., for the treatment of chronic kidney disease), patients undergoing immunosuppressive therapy, AIDS patients, diabetic patients, patients undergoing chemotherapy or radiotherapy for cancer, and immunocompromised states (resulting from genetic deficiencies, malnutrition, drug abuse, alcohol dependence, or other diseases or conditions that cause immunosuppression).

[0065] In certain embodiments, the immunocompromised subject is elderly. As humans and animals age, their immune response declines, and the robustness of the immune response is attenuated by the spread of low-affinity antibody responses. Thus, the subject in these embodiments can be a human patient over 45 years old, or over 50 years old. In some embodiments, the subject is a human patient 60 years old or older, 65 years old or older, or 70 years old or older.

[0066] In some embodiments, the subject is at risk of sepsis, severe sepsis, or septic shock due to nosocomial infection. Sepsis, severe sepsis, or septic shock due to nosocomial infection is sepsis, severe sepsis, or septic shock that develops during hospitalization. Because antibiotics are frequently used in hospitals, the microorganisms associated with sepsis, severe sepsis, or septic shock, and their resistance to antibiotics, may differ from those isolated outside the hospital.

[0067] In one aspect of the invention, a thymosin peptide regimen is administered to treat / prevent sepsis in patients at risk of sepsis. According to this aspect, a thymosin alpha peptide regimen can be used to stimulate the patient's immune system and provide a more rapid response to pathogen exposure (which can be predicted for the patient based on planned events in some embodiments), thereby preventing sepsis.

[0068] For example, the subject may be scheduled for invasive surgical procedures, and in these aspects, the thymosin alpha peptide regimen reduces the risk and / or severity of postoperative sepsis. Generally, invasive medical procedures have an infection risk, and exemplary procedures include joint replacement, organ or tissue transplantation or grafting, introduction of a prosthesis, removal of tissue including tumor or cancerous tissue, tonsillectomy, appendectomy, splenectomy, thymectomy, nephrectomy, amputation, removal of bone marrow, or other invasive medical procedures. In such aspects, the TA1 regimen may reduce the risk of sepsis.

[0069] In certain aspects, the patient may require the assistance of invasive medical devices, which cause exposure of the body to microorganisms and introduce an opportunistic environment for sepsis to occur. Thus, the devices can increase exposure to potential opportunistic bacteria and pathogens. Such devices include, but are not limited to, ventilators, urinary catheters, arterial catheters, feeding tubes, intravenous drips, stents, kidney dialysis, or artificial organs. In these aspects, the thymosin alpha peptide regimen helps to stimulate the patient's immune system to prevent or reduce the severity of any resulting sepsis, severe sepsis, or septic shock.

[0070] In certain embodiments, a patient is in need of, or is under the assistance of, a lung ventilation device, and the TA1 regimen serves to stimulate the patient's immune system and maintain the immune system in a stimulated state so as to reduce the risk or severity of ventilator-associated pneumonia. Ventilator-associated pneumonia (VAP) occurs in patients using a mechanical ventilator through an endotracheal tube or a tracheostomy tube and results from an infection in the alveoli. Pseudomonas aeruginosa is the most common Gram-negative bacterium causing VAP, and the genus Pseudomonas has natural resistance to many antibiotics. Other causative species for VAP include Klebsiella pneumoniae, which has natural resistance to some β-lactam antibiotics such as ampicillin and / or carbapenem, as well as cephalosporins and aztreonam. Serratia, Enterobacter, and Acinetobacter genera may also be associated with VAP and can also be resistant to antibiotics. In addition, the association between Staphylococcus aureus (including MRSA) and VAP is also increasing.

[0071] In some embodiments, a patient is undergoing, or scheduled to undergo, hemodialysis (e.g., due to chronic kidney disease). Since hemodialysis requires access to the circulatory system, patients undergoing hemodialysis may have their circulatory system exposed to microorganisms, which can lead to sepsis. Thus, in certain embodiments, the TA1 regimen described herein is initiated to prepare a patient for hemodialysis.

[0072] In some embodiments, the patient is a cancer patient and is undergoing or scheduled to undergo chemotherapy and / or radiation therapy, which often has an adverse effect on the patient's immune system. If the patient is undergoing or scheduled to undergo chemotherapy, the chemotherapy generally has a harmful effect on immune cells and, in particular, may include one or more alkylating agents (e.g., cisplatin, carboplatin, and ifosfamide), antimetabolites (5-fluorouracil or folic acid antagonists), topoisomerase inhibitors (e.g., camptothecin, etoposide), or taxanes (e.g., paclitaxel). In some embodiments, the thymosin α peptide regimen is administered prior to cancer therapy to stimulate the patient's immune system in order to prevent or reduce sepsis.

[0073] In one exemplary embodiment, the regimen of thymosin α peptides described herein is provided to leukemia patients and / or neutropenic patients, thereby preventing or reducing the severity of catheter-related sepsis, severe sepsis, or septic shock that may be caused by drug-resistant Staphylococcus aureus (e.g., MRSA and VRSA). In another exemplary embodiment, the thymosin α peptide regimen described herein is provided to bone marrow transplant patients, thereby preventing or reducing the severity of sepsis, such as that generally caused by Aspergillus, Candida, or CMV. In yet another embodiment, the regimen of thymosin α peptides described herein is provided to organ (e.g., kidney) transplant recipients, thereby preventing organ rejection that may result from CMV-based sepsis.

[0074] In certain embodiments, at the start of the TA1 regimen, symptoms of sepsis are absent or mild, but the presence of a microorganism or disease has been determined by culture, ELISA, or other diagnostic tests. In such embodiments, the thymosin alpha peptide regimen serves to stimulate the immune system to more rapidly develop an antibody response capable of dissipating the infection. In some embodiments, the thymosin alpha peptide regimen is an effective regimen provided concurrently with standard antibacterial, antiviral, or antifungal therapy.

[0075] A variety of diagnostic tests for sepsis or its related conditions are known in the art, and such tests are well known to those of ordinary skill in the art and can be used in the methods of the present invention. Such tests can include, but are not limited to, blood tests, other clinical tests, and imaging scans. Blood tests can include tests for evidence of infection (i.e., the presence of bacteria, fungi, or viruses), coagulation abnormalities, abnormal liver or kidney function, decreased oxygen utilization, electrolyte imbalances, decreased immune function (e.g., decreased monocyte HLA-DR levels), and other clinical tests. Other clinical tests can include urine tests (e.g., tests of urine for infectious pathogens), tests of wound secretions (e.g., tests of wound secretions for infectious pathogens), and tests of respiratory secretions (e.g., tests of respiratory secretions such as sputum mucus for infectious pathogens). Imaging tests can include X-rays (e.g., to visualize infections in the lungs), computed tomography (CT; e.g., to visualize infections in the appendix, pancreas, or intestine), ultrasound (e.g., to visualize infections in the bladder or ovaries), and magnetic resonance imaging (MRI; e.g., to identify soft tissue infections including abscesses within the spine), but are not limited thereto.

[0076] In certain embodiments, a patient (or a patient's sample, a sepsis-susceptible site, or an adjacent surrounding environment) is examined and found positive for the presence of one or more infectious organisms, including but not limited to Listeria, Pseudomonas (e.g., Pseudomonas aeruginosa), Serratia, Clostridium difficile, Staphylococcus aureus, Staphylococcus spp., Acinetobacter spp., Enterococcus spp., Enterobacter spp., Escherichia coli, Klebsiella spp., Streptococcus (e.g., Streptococcus pneumoniae), Haemophilus influenzae, and Neisseria meningitidis. In some embodiments, the infection involves drug-resistant or multi-drug resistant microorganisms such as Staphylococcus aureus, Enterococcus spp., Pseudomonas spp., Klebsiella spp., Escherichia coli, and / or Clostridium difficile, or the isolated bacterium is identified as such a drug-resistant or multi-drug resistant microorganism. In certain embodiments, the infectious pathogen is a drug-resistant Streptococcus pneumoniae, including penicillin resistance, methicillin resistance, and / or quinolone resistance (e.g., fluoroquinolone). In certain embodiments, the drug-resistant microorganism is methicillin-resistant or vancomycin-resistant Staphylococcus aureus (MRSA or VRSA) including moderately resistant isolates, or carbapenem-resistant Escherichia coli, Klebsiella, or Pseudomonas including moderately resistant isolates. The presence of such organisms can be determined or confirmed using diagnostic tests known in the art, or can be determined by a sudden increase in the occurrence of such infections in a medical institution.

[0077] In certain exemplary embodiments, the patient is a neutropenic patient suffering from an infection with Pseudomonas, Acinetobacter, or Escherichia coli, and the resulting sepsis, severe sepsis, or septic shock can be caused by drug-resistant microorganisms, or the patient is suffering from ventilator-associated pneumonia, which can involve an infection with Pseudomonas or Serratia, which can also result in drug-resistant sepsis, severe sepsis, or septic shock.

[0078] The regimen of thymosin alpha peptide may be administered concurrently with antibiotic therapy including beta-lactam antibiotics (e.g., methicillin, ampicillin, carbapenem, piperacillin); cephalosporins; fluoroquinolones (e.g., ciprofloxacin, levofloxacin, moxifloxacin), and / or macrolides (e.g., azithromycin, clarithromycin, dirithromycin, and erythromycin). The antibiotic therapy may be administered with additional therapeutic agents such as beta-lactamase inhibitors (tazobactam). In certain embodiments, thymosin alpha peptide reduces the duration of sepsis and the duration of treatment with the required antibiotics. In certain embodiments, the infection is determined to be resistant to such agents prior to initiating treatment with thymosin alpha peptide. In certain embodiments, the thymosin alpha peptide regimen is initiated or continued or repeated to help prevent recurrence after completion of antibiotic therapy after sepsis has clearly resolved. An effective regimen of thymosin alpha peptide (e.g., 1, 2, 3, 4, 5 or 6 doses) may cover the entire course of antibacterial therapy and may provide additional immunity in the immune response over the entire period.

[0079] In certain embodiments, the patient has sepsis resulting from a viral infection selected from cytomegalovirus (CMV), RSV, influenza virus, herpes simplex virus type 1, and parainfluenza virus. The thymosin alpha peptide regimen described herein may reduce the severity and / or duration of virus-based sepsis, severe sepsis or septic shock and may be provided in combination with appropriate antiviral therapy which may be a virus-neutralizing antibody or a small molecule inhibitor such as Tamiflu. In certain embodiments, the thymosin alpha peptide regimen is initiated or continued or repeated to help prevent recurrence after completion of other therapies after virus-based sepsis, severe sepsis or septic shock has clearly resolved.

[0080] In yet other embodiments, the patient has sepsis resulting from a fungal infection of Aspergillus spp. (e.g., Aspergillus fumigatus) or Candida spp. (e.g., Candida albicans), which may also show resistance to treatment with antibiotics. In certain embodiments, the thymosin peptide regimen is administered in conjunction with an antifungal treatment. Antifungal therapies include azole drugs such as imidazoles (e.g., ketoconazole) or triazoles (e.g., fluconazole). In certain embodiments, the thymosin alpha peptide regimen is initiated or continued or repeated after the infection has clearly resolved to help prevent recurrence after antifungal treatment.

[0081] According to some embodiments of the invention, administration of thymosin alpha peptide by the methods of the invention provides a statistically significant treatment effect. In one embodiment, the statistically significant treatment effect is determined based on one or more standards or criteria provided by one or more regulatory authorities in the United States, such as the FDA, or in other countries. In another embodiment, the statistically significant treatment effect is determined based on results obtained from the settings and / or procedures of clinical trials approved by regulatory authorities.

[0082] In some embodiments, the statistically significant treatment effect is determined based on a patient population of at least 300, 400, 500, 600, 700, 800, 900, 1000 or 2000 patients. In some embodiments, the statistically significant treatment effect is determined based on data obtained from the setting of a randomized double-blind clinical trial. In some embodiments, the statistically significant treatment effect is determined based on data having a p-value of about 0.05, 0.04, 0.03, 0.02 or 0.01 or less. In some embodiments, the statistically significant treatment effect is determined based on data having a confidence interval of 95%, 96%, 97%, 98% or 99% or more. In some embodiments, the statistically significant treatment effect is determined based on the approval of a Phase III clinical trial of the method provided by the invention, for example, by the FDA in the United States.

[0083] In some embodiments, a statistically significant treatment effect is determined by a randomized, double-blind clinical trial of at least 300 or 350 patients treated with thymosin α peptide, combined with standard treatment but not combined with any protease inhibitor. In some embodiments, a statistically significant treatment effect is determined by a randomized clinical trial of at least 300 or 350 patients and by using 28-day mortality, in-hospital mortality, in-ICU mortality, ICU length of stay, ICU-free days, sequential organ failure assessment (SOFA), relative risk of death, ICU frequency, duration of mechanical ventilation, frequency of mechanical ventilation, mechanical ventilation-free days, HLA-DR expression, or any combination thereof, or any other generally approved criteria for sepsis assessment.

[0084] Generally, statistical analysis can include any appropriate method approved by a regulatory authority, such as the US FDA or China or any other country. In some embodiments, statistical analysis includes unstratified analysis, such as log-rank analysis based on the Kaplan-Meier method, Jacobson-Truax method, Gulliken-Lord-Novick method, Edwards-Nunnally method, Hageman-Arrindel method, and Hierarchical Linear Modeling (HLM), as well as Cox regression analysis.

[0085] In some embodiments, biomarkers of sepsis, severe sepsis or septic shock can be used to predict treatment response and / or determine treatment efficacy. In some embodiments, mHLA-DR can be measured and an improvement in the mHLA-DR level can be used as an indicator of a favorable treatment response. In some embodiments, a decreased or reduced level of the mHLA-DR biomarker (including a decrease in protein expression level, mRNA transcription level, number of mHLA-DR positive monocytes) that increases after administration of thymosin alpha peptide is predictive of treatment response. In some embodiments, this information can be used in determining a treatment regimen (as described herein) for the treatment of sepsis, severe sepsis or septic shock using the thymosin alpha peptide of the invention. Accordingly, the present invention provides a method for determining a treatment regimen, comprising detecting an increase or decrease in the level of a biomarker of sepsis, severe sepsis or septic shock in a biological sample from a subject treated with thymosin alpha peptide, and determining a treatment regimen for the thymosin alpha peptide based on the increase or decrease in the level of one or more biomarkers of sepsis, severe sepsis or septic shock in the biological sample. In some embodiments, the biomarker of sepsis, severe sepsis or septic shock is mHLA-DR. In some embodiments, a decreased or reduced level of mHLA-DR is an indicator of treatment response and / or treatment efficacy of treatment with thymosin alpha for sepsis, severe sepsis or septic shock. In some embodiments, an increase or enhancement of a decreased or reduced level of mHLA-DR in response to treatment with thymosin alpha peptide is an indicator of treatment response and / or treatment efficacy of treatment with thymosin alpha peptide for sepsis, severe sepsis or septic shock. In some embodiments, recovery of the mHLA-DR level to a pre-determined standard level is an indicator of a better treatment prognosis (e.g., better survival rate) in treatment with thymosin alpha peptide.In some embodiments, a higher elevation of the mHLA-DR level is an indicator of a better treatment prognosis in treatment with thymosin α peptide.

[0086] As used herein, the expression "determining treatment efficacy" and variations thereof can include any method for determining whether a treatment confers a benefit on a subject. The term "treatment efficacy" and variations thereof are generally indicated by the reduction of one or more signs or symptoms associated with a disease and can be readily determined by one of ordinary skill in the art as the reduction of one or more signs or symptoms of the indication or disease being treated. "Treatment efficacy" can also refer to the prevention or remission of signs and symptoms of toxicity typically associated with standard treatments for a disease, i.e., chemotherapy or radiation therapy for the treatment of cancer. Such methods are specific to the indication and disease and can include any method well known in the art for determining that a treatment is having a beneficial effect on a patient. For example, evidence of prevention, treatment, or reduction of sepsis, severe sepsis, or septic shock, and determination of treatment response can include a decrease in the incidence of sepsis, severe sepsis, or septic shock, as well as a decrease in in-hospital mortality, a decrease in ICU mortality, an increase in the number of days not requiring ICU, a decrease in the length of stay in the ICU, a decrease in the duration of the ICU stay, a decrease in the frequency of the ICU stay, a beneficial or improved sequential organ failure assessment score (SOFA), a decrease in the duration of mechanical ventilation, and a decrease in the frequency of mechanical ventilation. Treatment efficacy can include, but is not limited to, remission of sepsis, severe sepsis, or septic shock, including a decrease or reduction in the underlying infection causing sepsis, severe sepsis, or septic shock.Furthermore, the treatment efficacy can also include, but is not limited to, an overall improvement in the subject's overall health, such as an improvement in the patient's quality of life, an increase in the predicted survival rate of the subject, a decrease in depressive states, or a decrease in the recurrence rate of symptoms (increase in remission time) (see, for example, Physicians' Desk Reference (2010) and Dellinger R.P., et al., Surviving Sepsis Campaign: international guidelines for management of severe sepsis and septic shock: 2008; Intensive Care Med. 34(4):783-785, (2008)).

[0087] Predetermined standard levels of biomarkers for sepsis, severe sepsis, or septic shock can be defined using various methods known to those of skill in the art. Generally, the standard level of a biomarker is determined by determining the level of the biomarker in a sufficiently large number of samples obtained from subjects that are healthy controls (e.g., subjects not exhibiting sepsis, severe sepsis, or septic shock). Additionally, information on standard levels can be obtained from publicly available databases and other sources. (See, e.g., Bunk, D.M., "Reference Materials and Reference Measurement Procedures: An Overview from a National Metrology Institute," Clin. Biochem. Rev., 28(4):131-137 (2007); Suraj Peri1, et al., "Development of Human Protein Reference Database as an Initial Platform for Approaching Systems Biology in Humans" Genome Res. 13: 2363-2371 (2003); Remington: The Science and Practice of Pharmacy, Twenty First Edition (2005)). In some embodiments, an increase or decrease in the level of one or more markers of sepsis, severe sepsis, or septic shock in a sample obtained from a subject treated with thymosin alpha peptide is determined by comparing the level of one or more biomarkers of sepsis, severe sepsis, or septic shock to a predetermined standard level.

[0088] Determining the treatment efficacy of treatment with thymosin alpha peptide and adjusting the treatment regimen for treatment with thymosin alpha peptide using information regarding an increase or decrease in the level of one or more biomarkers of sepsis, severe sepsis, or septic shock. In some embodiments, the treatment efficacy can be used to determine whether to continue treatment with thymosin alpha peptide. In other embodiments, the treatment efficacy can be used to determine whether to discontinue treatment with thymosin alpha peptide. In other embodiments, the treatment efficacy can be used to determine whether to modify the treatment with thymosin alpha peptide. In other embodiments, the treatment efficacy can be used to determine whether to increase or decrease the dosage of thymosin alpha peptide administered. In other embodiments, the treatment efficacy can be used to determine whether to change the dosing frequency. In further embodiments, the treatment efficacy can be used to determine whether to change the number of doses per day, the number of doses per week, or the number of times per day. In still further embodiments, the treatment efficacy can be used to determine whether to change the dosage amount.

[0089] Methods for obtaining biological samples are well known in the art and any standard method for obtaining a biological sample can be used. Biological samples useful in the methods of the present invention include, but are not limited to, serum, blood, plasma, whole blood and derivatives thereof, skin, hair, hair follicles, saliva, oral mucus, vaginal mucus, sweat, tear fluid, epithelial tissue, urine, semen, seminal fluid, seminal plasma, prostatic fluid, bulbourethral fluid (Cowper's fluid), excrement, biopsy, ascites, cerebrospinal fluid, lymph fluid, and tissue extract samples or biopsies (see, for example, Clinical Proteomics: Methods and Protocols, Vol. 428 in Methods in Molecular Biology, Ed. Antonia Vlahou (2008)).

[0090] All publications mentioned and cited in this specification are hereby incorporated by reference in their entirety. It is understood that the disclosed invention is not limited to the specific methods, protocols, and materials described, as these may vary. Also, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is understood to be limited only by the appended claims.

[0091] One of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by the appended claims.

Examples

[0092] Example 1: Efficacy of thymosin α1 for severe sepsis (ETASS): A multicenter, combined, simple, blinded, randomized, controlled trial Overview Introduction: Severe sepsis is associated with high mortality despite the implementation of guideline recommendations. Adjunctive treatments may be effective and warrant further investigation. Considering the important role of immunological derangement in severe sepsis, thymosin α1 (Tα1) is thought to be a promising and beneficial immunomodulatory agent. The trial assesses whether Tα1 improves 28-day overall mortality and immune function in patients with severe sepsis. Methods: We conducted a multicenter, randomized, controlled trial in six tertiary teaching hospitals in China from May 12, 2008, to December 22, 2010. Eligible patients admitted to the ICU with severe sepsis were randomly assigned by a central randomization center to a control group or a Tα1 group (1:1 ratio). The primary endpoint was death from any cause, evaluated 28 days after enrollment. Secondary endpoints included the dynamic changes in sequential organ failure assessment (SOFA) and mHLA-DR (human leukocyte antigen DR on monocytes) on days 0, 3, and 7 in both groups. All analyses were performed on an intention-to-treat basis. Results: 361 patients were assigned to either the control group (n = 180) or the Tα1 group (n = 181). Mortality from any cause within 28 days in the Tα1 group and the control group was 26.0% and 35.0%, respectively, at the marginal p-value (unstratified analysis, p = 0.062; log-rank, p = 0.049); the relative risk of death in the Tα1 group compared with the control group was 0.74 (95% CI 0.54 to 1.02). A greater improvement in mHLA-DR was observed in the Tα1 group compared with the control group on day 3 (mean difference in mHLA-DR change between the two groups was 3.9%, 95% CI 0.2 to 7.6%, p = 0.037) and day 7 (mean difference in mHLA-DR change between the two groups was 5.8%, 95% CI 1.0 to 10.5%, p = 0.017). No serious drug-related adverse events were recorded.

[0093] Conclusion: The use of Tα1 therapy in combination with conventional medical therapy may be effective in improving clinical outcomes in the target population of severe sepsis.

[0094] Trial registration: ClinicalTrials.gov, NCT00711620.

[0095] Introduction Severe sepsis is an important cause of admission to intensive care units (ICUs) worldwide, which is characterized by high adult mortality [1-3]. More than 750,000 people are diagnosed with severe sepsis annually in the United States, and 215,000 of them die [3]. The reported mortality rate of severe sepsis is 28% to 35.5% [3-7]. Despite the adoption of treatment bundles based on the Surviving Sepsis Campaign (SSC) guidelines, the mortality rate is reported to be approximately 30% [4]. The important role of immunological disturbances in the course and poor prognosis has increased interest in immunotherapy [8, 9]. Thymosin alpha 1 (Tα1) is a natural thymosin peptide first described and characterized by Goldstein et al.

[10] . It acts as an endogenous regulator of both the innate and adaptive immune systems

[11] . It has been used worldwide to treat diseases associated with immune dysfunction, including viral infections such as hepatitis B and C, certain cancers, and for vaccine enhancement [12, 13]. In particular, recent developments in immunomodulation research have shown the beneficial effects of treatment with Tα1 in sepsis patients. However, the results of these studies should be carefully considered because of the small sampling size and the use of more than one drug as a therapeutic intervention [14-16]. This multicenter, randomized, controlled trial was conducted to determine the efficacy of Tα1 in the treatment of severe sepsis.

[0096] Materials and methods The inventors conducted a prospective, controlled, single-blind, multi-center, randomized clinical trial, which was carried out in the ICUs of six tertiary teaching hospitals. The Ethics Committee of the First Affiliated Hospital of Sun Yat-sen University approved the protocol (200815). Written informed consent was obtained from the patients or, for those who were unable to consent, from their next of kin. The trial was registered at ClinicalTrials.gov, NCT 00711620.

[0097] Patients Patients diagnosed with severe sepsis who were admitted to the ICU between May 12, 2008 and December 22, 2010 were enrolled in the trial. The criteria for severe sepsis were a modified version of those defined by Bernard et al. (see Supplementary File 1) [7]. Patients were eligible for inclusion in the trial if, at the time of screening, they were known or suspected to be infected based on clinical data, and if they had two or more signs of systemic inflammation and dysfunction of at least one organ or organ system induced by sepsis.

[0098] Randomization and masking To minimize the heterogeneity of severe sepsis and the impact of variability between the hospitals where patients were recruited on the results, stratification by study center, combined with computer-generated block randomization (block size = 8), was used in the enrollment process in the order of adoption. The method of randomization and the block size were blinded until the data analysis was completely finished. The clinicians who enrolled the subjects were not involved in data collection. Eligible patients were randomly assigned at a 1:1 ratio to each hospital such that, after telephone confirmation through the randomization center, 4 people in each block received the test drug and the other 4 were assigned to the control group. The assignment order was kept confidential from the researchers. To prevent prior knowledge of treatment assignment and disruption of the assignment order, case report forms (CRFs) were filled out on the trial registration sheets, informed consent was obtained, and then the unique participation number and the assigned group were disclosed; the unique numbers created could not be changed or deleted later. The inventors used normal saline as a placebo. The patients were blinded to treatment assignment. All statistical analyses were performed while maintaining masking.

[0099] Administration of the Test Drug and Management of Sepsis In the Tα1 group, patients received subcutaneous injections of 1.6 mg of Tα1 (ZADAXIN®, SciClone Pharmaceuticals, Foster City, CA, USA) twice a day for 5 consecutive days and then once a day for 2 consecutive days. Prior to administration, the lyophilized powder was to be reconstituted with 1 ml of the provided diluent (sterile water for injection). After reconstitution, the final concentration of Tα1 was 1.6 mg / mL. In the control group, patients received subcutaneous injections of 1 mL of normal saline twice a day for 5 consecutive days and then once a day for 2 consecutive days. According to the trial protocol, treatment had to be initiated within 4 hours after enrollment.

[0100] The treating physician instructed the care of the patient in accordance with current international guidelines

[17] , including appropriate empirical antibiotic therapy based on current recommendations, mechanical ventilation regimen (pressure control mode), blood glucose control, cardiopulmonary resuscitation and hemodynamic support, organ support, sedation or analgesia if necessary, and appropriate nutritional regimens. Empirical antibiotic therapy was considered appropriate if at least one effective drug was included in the empirical antibiotic therapy within the first 24 hours of admission to the ICU, and the optimal dose and correct route of administration were in accordance with medical standards, and there were no microorganisms detected microbiologically in the bloodstream or lesions among the ICU survivors. If empirical antibiotic therapy had to be changed after the detection of microbiological microorganisms, it was considered inappropriate, while in the case of non-survivors without microorganisms detected microbiologically in the bloodstream or lesions, it was considered inevaluable [18 - 20].

[0101] Outcome and data collection The primary efficacy criterion was death from any cause, which was evaluated 28 days after the start of treatment assignment. Secondary criteria included the dynamic changes in Sequential Organ Failure Assessment (SOFA), CD4+ / CD8+ and monocyte human leukocyte antigen DR (mHLA-DR) expression measured on day 0 (registration day), day 3 and day 7 in both groups. All mHLA-DR measurements were performed in the central laboratory of the First Affiliated Hospital of Sun Yat-sen University. 1 ml of EDTA-untreated whole blood was stored on ice immediately after collection and transferred to the central laboratory as soon as possible to ensure measurement within 3 hours after collection. The method for measuring mHLA-DR was also described in our previous paper

[21] . Once a patient was registered, data including demographic characteristics, microbiological findings (main source of infection and identified microorganisms) and complications were collected if available. The following clinical parameters were recorded on specific days after registration: on day 0, severity as evaluated by Acute Physiology and Chronic Health Evaluation II (APACHE II); on days 0, 3, 7, SOFA, hematological and biochemical findings, mHLA-DR, CD4+ / CD8+ test results. The time of the first organ dysfunction was retrospectively estimated according to objective data such as blood gas analysis at the time the patient was registered.

[0102] Statistical analysis and sample size Based on previous studies

[22] , a sample size of 334 patients was required to show a decrease in 28-day mortality from 50% to 35% with treatment by Tα1 using a two-sided test (error = 5%; power = 80%). Assuming an expected dropout rate of 10%, the trial would require the enrollment of 368 patients in total. Demographic data, outcome data, and other clinical parameters were summarized for continuous variables by the median with category variable frequencies, and for mean values ± standard deviation (SD) or interquartile range (IQR). Ratios were compared using the chi-square test or Fisher's exact test. Continuous variables were tested using a t-test for those with a normal distribution or the Wilcoxon rank-sum test for those with a non-normal distribution. Comparison of primary endpoints between two groups was performed using the Cochran-Mantel-Haenszel test, with patients stratified by a number of baseline covariates such as mHLA-DR, APACHE, and SOFA scores, surgical and cancer histories, gender, and age. The corresponding relative risk (RR) with 95% confidence interval (CI) was calculated using the logit adjustment method. The Kaplan-Meier estimator without adjustment for baseline covariates was used for survival time analysis, and the log-rank test was used for comparison. To estimate the mean change from baseline of clinical parameters, a linear mixed model for repeated measures was used, taking into account center-based clustering and repeated measures within patients. This model included terms for baseline measurement, treatment group, visit, and the interaction of treatment and visit. The least-squares mean with 95% CI was reported. The inventors also analyzed the efficacy parameters of the test drug in various pre-specified subgroups. The heterogeneity of treatment effects between subgroups was evaluated using a test for interaction. Consistent with the Intention-to-treat principle, all analyses were based on all available populations consisting of the population with baseline and the population with at least one post-baseline efficacy measurement, without hypothesizing or attributing missing data. All statistical analyses were performed using SAS software (SAS 9.1.3; SAS Institute Inc., Cary, NC, USA).The P-values on both sides were reported, and a P-value less than 0.05 was considered statistically significant.

[0103] Results Test profile From May 12, 2008 to December 22, 2010, 367 eligible patients were randomized (Figure 1). In the Tα1 group, 2 patients were excluded: in one patient, consent was withdrawn after being diagnosed with typhoid and was immediately transferred to an infectious disease hospital; in the other case, consent was withdrawn before the infusion. In the control group, consent was withdrawn after registration in 4 cases. A total of 361 randomized patients were followed up for the entire 28-day study period without dropout. Among the 181 patients in the Tα1 group, 162 patients completed the test in accordance with the protocol regarding the use of the drug, while the other 19 patients received at least 1.6 mg of Tα1 but were transferred out of the ICU, so the treatment did not fully comply with the protocol.

[0104] Baseline data Both groups had similar characteristics in most demographic and baseline variables (Table 1), but the patients in the Tα1 group had a longer period from the time of the first observed organ dysfunction to the time of registration (42 hours vs. 28 hours, P = 0.003). Almost 80% of the patients had at least two dysfunctional organs at the time of registration. The lungs and cardiovascular system were the most frequently affected organ systems, with incidence rates of 94.7% and 65.7% respectively. The most common infection sites were the lungs and abdomen, with incidence rates of 74.5% and 27.4%, and mixed pathogens or Gram-negative bacteria accounted for the majority of cases. There was no difference in the treatment with appropriate antibiotics (see Table 2). The baseline of the clinical data between the two groups was comparable, as shown in Table 3. The patients in the Tα1 group had a lower level of mHLA-DR (47.1% vs. 58.0% in the control group, P = 0.02), but the distribution of each stratum in the two groups was similar.

[0105] (Table 1) Baseline characteristics in both study groups TIFF2025107346000002.tif34170TIFF2025107346000003.tif215170TIFF2025107346000004.tif122170APACHE II, Acute Physiology and Chronic Health Evaluation II; BMI, Body Mass Index; COPD, Chronic Obstructive Pulmonary Disease; IQR, Interquartile Range; SOFA, Sequential Organ Failure Assessment; Tα1, Thymosin α1; years old, age.

[0106] (Table 2) Infection sites, causes of infection, and treatment with appropriate antibiotics in patients with severe sepsis TIFF2025107346000005.tif50170TIFF2025107346000006.tif214170 * Patients may have more than one infection site; † Other infection sites included the skin, central nervous system, bone, and joints; ‡ Patients may have more than one cultured organism. Tα1, Thymosin α1.

[0107] (Table 3) Baseline levels of clinical values TIFF2025107346000007.tif161170CD, Cluster of Differentiation; CI, Confidence Interval; mHLA-DR, Monocyte Human Leukocyte Antigen DR; SOFA, Sequential Organ Failure Assessment; Tα1, Thymosin α1.

[0108] Test results Primary evaluation criteria Within 28 days after registration, 47 out of 181 patients (26.0%) in the Tα1 group and 63 out of 180 patients (35.0%) in the control group took a breath. The relative risk of death in the Tα1 group was 0.74 (95% CI 0.54 - 1.02) with a P-value of 0.062 in the unstratified analysis compared to the control group. In the Tα1 group, the mortality rate decreased absolutely by 9.0% (95% CI -0.5 - 18.5%). The survival time and onset curves of the two groups are presented in Figure 2. Patients in the Tα1 group survived longer after registration than the control group (log-rank, P = 0.049). A total of 52 out of 181 patients (28.7%) in the Tα1 group and 71 out of 180 patients (39.4%) in the control group died in the hospital. The relative risk of death in the hospital in the Tα1 group was 0.73 (95% CI 0.54 - 0.98) compared to the control group, and the P-value was 0.032. There were no significant differences between the two groups in ICU mortality, days without mechanical ventilation, days without ICU stay, length of ICU admission, and duration of mechanical ventilation (Table 4).

[0109] (Table 4) Primary endpoints and prognosis TIFF2025107346000008.tif88170 The "days without need" were calculated as the number of days the patient survived during the 28-day study period and did not require a given endpoint (mechanical ventilation and ICU stay). CI, confidence interval; IQR, interquartile range, Tα1, thymosin α1.

[0110] Secondary endpoints The dynamic changes in SOFA and clinical measurements are summarized in Table 5. A persistent increase in the mHLA-DR value (% of positive monocytes) was observed in both groups. The mean changes from baseline on day 3 and day 7 were 4.1% and 11.2% in the control group, and 8.0% and 17.0% in the Tα1 group. Patients in the Tα1 group had a lower mHLA-DR baseline than the control group on day 0. Greater improvement in mHLA-DR was observed in patients in the Tα1 group on day 3 (the mean difference in the change in mHLA-DR between the two groups was 3.9%, 95% CI 0.2 - 7.6%, P = 0.037) and day 7 (the mean difference in the change in mHLA-DR between the two groups was 5.8%, 95% CI 1.0 - 10.5%, P = 0.017). The mean changes in the SOFA score on day 3 and day 7 were -1.3 (95% CI -1.7 - -0.8, P < 0.001) and -1.8 (95% CI -2.4 - -1.3, P < 0.001) in the control group, and -1.8 (95% CI -2.3 - -1.4, P < 0.001) and -2.5 (95% CI -3.1 - -2.0, P < 0.001) in the Tα1 group. The tendency for a decrease in the SOFA score within 7 days seemed to be smooth in the Tα1 group. The CD4+ / CD8+ ratio remained unchanged in both groups over 7 days.

[0111] (Table 5) Dynamic changes in SOFA and clinical measurements TIFF2025107346000009.tif146159 * ΔDay 3 and ΔDay 7 were defined as the numerical changes on day 3 and day 7 compared to day 0. a P < 0.05; b P < 0.01. CD, surface antigen classification; CI, confidence interval; mHLA-DR, human leukocyte antigen DR of monocytes; SOFA, sequential organ failure assessment; Tα1, thymosin α1.

[0112] Subgroup analysis The mortality rates among patients in a pre-specified subgroup are shown in Figure 3. In a pre-specified analysis of the primary endpoint (patients were stratified according to APACHE II score, SOFA score, mHLA-DR level, surgical or cancer history, gender, and age), Tα1 tended to improve the outcomes but was not statistically significant. In the subgroup analysis of patients with cancer, the relative risk of death in the Tα1 group compared to the control group was 0.46 (95% CI 0.25 - 0.86, P = 0.01); while in patients without cancer, the relative risk of death in the Tα1 group was 0.91 (P = 0.07 by test for interaction).

[0113] Adverse event The safety and tolerability assessment of Tα1 (see Additional file 3) was based on a comparison of all available information obtained from the two groups with respect to outliers detected in clinical safety data, serious adverse events related to the drug (evaluated by the investigators), and deterioration of the function of organs and organ systems (evaluated by scoring of individual SOFA components for the respiratory, cardiovascular, hepatic, coagulation, renal, and nervous systems that occurred during the treatment).

[0114] In this study, no serious adverse events (SAEs) related to Tα1 were reported, and no treatment was discontinued due to intolerance or adverse events. There was no statistical difference between the control group and the Tα1 group with respect to the frequency of deviations from clinical values and organ or organ system disorders due to any cause (see Table 6).

[0115] (Table 6) Frequencies of patients with outliers in clinical safety assays and organ and organ system disorders due to any cause TIFF2025107346000010.tif108170 * Organ and organ system disorders based on deterioration of SOFA component scores during treatment. ALT, alanine aminotransferase; AST, aspartate aminotransferase; SOFA, Sequential Organ Failure Assessment; Tα1, thymosin α1.

[0116] Investigation Abnormal regulation of the immune system plays an important role in the course of sepsis. Previously, it was thought that the exacerbated inflammatory response and the accompanying organ damage induced by inflammation were the main causes leading to death in sepsis. However, recent studies have shown that there is heterogeneity in the immune responses of patients with sepsis, with some patients appearing to be immune-stimulated while others appear to be suppressed

[23] . Both pro-inflammatory and anti-inflammatory drugs have been evaluated, but few have been found to significantly reduce mortality [24-26]. Tα1 is thought to have an immunomodulatory effect mainly by influencing the enhancement of T cell function [27, 28]. Tα1 has also shown an effect that exceeds its effect on T lymphocytes by acting as an endogenous regulator of both the innate and adaptive immune systems [11, 29]. Tα1 plays a unique role in achieving the balance between pro-inflammatory cytokine production and anti-inflammatory cytokine production through the involvement of individual Toll-like receptors (TLRs) that act on various dendritic cell (DC) subsets and the involvement of the MyD88-dependent signaling pathway. Tα1 can increase the secretion of IL-12, IL-2, IFN-α and IFN-γ to present an antimicrobial effect, and can increase the proportion of IL-10 and regulatory T cells (Tregs) to control inflammation [11, 30-32]. Therefore, it is an appropriate immunomodulatory factor for treating severe sepsis characterized by significant heterogeneity in immune function.

[0117] Our data suggest that administration of Tα1 decreased 28-day mortality from any cause by 9.0% (marginal P-value, non-stratified analysis P = 0.062; log-rank, P = 0.049) and decreased in-hospital mortality (P = 0.032) in patients clinically diagnosed with severe sepsis. As shown in our previous trials and another epidemiological study on severe sepsis in China, our study was designed to detect an absolute mortality reduction from a projected 50% to 15% in the future [22, 33]. The size of the mortality and drug effect did not match our expectations (which could reach the marginal p-value) in the comparison of 28-day survival between the two groups. In contrast to our results, previous trials in adults have shown that Tα1 significantly decreased mortality from 13.1% to 18% compared to the control group [14-16]. The following reasons may explain this discrepancy in different trials. First, heterogeneity of the patient population and different treatment approaches may have affected the outcomes; second, previous trials have not reported allocation concealment, which may have had an unexpected impact on the results. Schulz et al. showed that the odds ratio deteriorated by 41% in inappropriately concealed trials and 30% in obscurely concealed trials

[34] . Third, such trials used more than one drug as a therapeutic intervention, making it difficult to attribute the beneficial effects observed for each agent.

[0118] The biomarker most frequently evaluated to assess the immune function in severe sepsis is mHLA-DR. It seems generally agreed that the loss of mHLA-DR is a reliable marker for the occurrence of immune dysfunction in patients with severe sepsis [35, 36]. Recent studies have shown that the dynamic changes of mHLA-DR over time are better predictors of mortality and that the recovery of mHLA-DR is associated with a better prognosis [21, 37, 38]. In this study, a greater improvement in mHLA-DR was observed in the Tα1 group than in the control group on days 3 and 7, suggesting that Tα1 may improve the immune function in severe sepsis. The CD4+ / CD8+ ratio is another parameter for assessing the immune status in sepsis. A decreased CD4+ / CD8+ ratio has been associated with the occurrence of severe sepsis and multiple organ failure (MOF) in trauma patients

[39] . Some studies have shown that thymosin α1 can increase the CD4+ / CD8+ ratio [40, 41]. In contrast, one observational study showed that mHLA-DR, rather than CD4+, CD8+ or the CD4+ / CD8+ ratio, can predict the prognosis of severe sepsis

[42] . In the study by the present inventors, no statistically significant difference in the CD4+ / CD8+ ratio was found between the two groups. The tendency of a decrease in the SOFA score within 7 days seemed to be smooth in the Tα1 group, but there was no significant difference in the change between the two groups. However, considering the fact that the present inventors only observed the changes in these indices for only 7 days, there may be some differences between the two groups if the observation is extended to 14 days or 28 days.

[0119] The median time from the detection of the first organ dysfunction to registration exceeded 24 hours in both groups, but was longer in the Tα1 group. The inventors adopted a retrospective method to determine the time window from the onset of the detected first organ dysfunction to registration in the study according to objective data (such as blood gas analysis), and much of those data were obtained before transferring critically ill septic patients to the ICU [7]. However, patients without the presented objective data may also be suffering from severe sepsis, and the delay in clinical examination may have substantially underestimated the time after onset. In other words, the time after onset determined by clinical examination in departments other than the ICU was uncontrollable for the inventors, and errors were likely to occur especially when the estimation was based on hours rather than days. The exact time window from the onset of the first organ dysfunction to registration may have exceeded the recorded time and may have been balanced between the two groups. A better way to enroll critically ill septic patients in immunotherapy studies may be through mHLA-DR values, which have been proven to be good predictors for evaluating the immune status of patients and good parameters for individualized goal-directed therapy

[43] .

[0120] A reduction in relative risk of death was observed in all subgroups, including those stratified according to age, sex, APACHE II score, SOFA score, and mHLA-DR level, but was not statistically significant. The purpose of analyzing different predefined subgroups in the inventors' study was to prepare for their future studies in specific groups of targeted critically ill septic patients who may benefit from Tα1 treatment. The results of subgroup analysis in the inventors' study were inconclusive, and whether Tα1 is more effective in specific groups of critically ill septic patients and this should be explored in a larger-scale trial with a larger sampling size.

[0121] The types of pathogens and empirical antibiotic therapy are very important factors that affect the outcomes of severe sepsis. It should be noted that the microbial origins are substantially diverse in different regions and even in different hospitals within the same region. Empirical therapies are also diverse. In this study, the isolation rates of Gram-negative bacteria (Pseudomonas, Acinetobacter) were higher compared to some other infection epidemiological studies in the ICU

[44] . In fact, the relatively higher incidence of Pseudomonas and Acinetobacter infections is not abnormal in China

[33] , so appropriate empirical therapies are adjusted accordingly.

[0122] Thymosin α1 has been shown to be a safe and well-tolerated agent in other studies [12, 13]. No serious adverse events were observed in the tests by the inventors. Abnormal clinical values and organ and organ system disorders due to any cause were similar in both groups. However, subjective sensations such as irritation or burns, systemic diseases or digestive diseases were difficult to evaluate in patients with severe sepsis due to the severity of the disease, sedation or anesthesia.

[0123] In the studies of the present inventors, several factors limit the scope within which the results can be generalized. First, the test population was heterogeneous with respect to clinical characteristics. Although more than 80 baseline characteristics were equivalent between the two groups, there were differences in mHLA-DR expression, which was probably due to patient heterogeneity and relatively small sampling sizes. In fact, unbalanced baseline characteristics between groups were not rare even in large samples in severe sepsis trials [45, 46]. In the studies of the present inventors, a linear mixed model for long-term data was applied with adjustment for baseline values to evaluate whether outcomes differed by treatment group. This method is widely used in multi-center studies [47, 48]. Second, considering the heterogeneity of severe sepsis, a certain patient group might have benefited more from the intervention than other sepsis patients. Future individualized and targeted treatment of severe sepsis with Tα1 should be implemented in specific patient groups targeted. One biomarker that can be used to stratify patients by their immune status is mHLA-DR. Meisel et al. reported that mHLA-DR levels were associated with immunosuppression in sepsis patients who benefited from treatment with granulocyte macrophage colony-stimulating factor (GM-CSF)

[43] . The present inventors will attempt to recruit mHLA-DR-targeted immunosuppressed patients in future studies. Third, since a significant proportion of patients were discharged from the ICU within one week, it was difficult to ensure that complete clinical and follow-up data could be obtained, and the present inventors simply collected clinical data within 7 days and followed up the survival status at 28 days. Collection of more extensive clinical data and a longer follow-up period would probably provide more important information. Fourth, there are few biomarkers for assessing immunological abnormalities. In this trial, the present inventors adopted the widely used mHLA-DR. Fifth, from the trials of the present inventors, extension of treatment beyond 7 days or an increase in dose would probably result in a significant improvement in the outcomes of severe sepsis patients. Sixth, since the present inventors could not obtain an identical placebo, they did not use a double-blind method and were only blinded for the patients and the statisticians.To minimize potential bias, randomization and appropriate allocation concealment were taken care of in the trial

[34] , and the primary and secondary evaluation criteria were rather objective than subjective.

[0124] Considering these limitations, this study is a preliminary investigation of the efficacy of thymosin α1 in severe sepsis, and further double-blind trials are needed.

[0125] Conclusion This RCT demonstrates that thymosin α1 therapy in combination with conventional medical therapy can be effective in improving clinical outcomes in a standardized population of severe sepsis. The need for larger multi-center studies to confirm these findings is indicated.

[0126] Considering the important role of immunological abnormalities in severe sepsis, immunotherapy can be an important adjuvant treatment.

[0127] This study demonstrates that immunomodulation with thymosin α1 can effectively improve the outcomes of patients with severe sepsis. A beneficial effect on the immune function of patients with severe sepsis was also observed. Further studies are needed to confirm these findings.

[0128] References TIFF2025107346000011.tif139156TIFF2025107346000012.tif222157TIFF2025107346000013.tif217156TIFF2025107346000014.tif213156TIFF2025107346000015.tif225156TIFF2025107346000016.tif225156TIFF2025107346000017.tif209157

[0129] Sequence Information SEQUENCE LISTING <110> SciClone Pharmaceuticals, Inc. First Affiliated Hospital, Sun Yat-sen University <120> USE OF THYMOSIN ALPHA FOR THE TREATMENT OF SEPSIS <150> US 61 / 618,563 <151> 2012-03-30 <150> US 61 / 643,824 <151> 2012-05-07 <150> US 13 / 835,107 <151> 2013-03-15 <160> 1 <170> PatentIn version 3.5 <210> 1 <211> 28 <212> PRT <213> Artificial Sequence <220> <223> THYMOSIN ALPHA POLYPEPTIDE <400> 1 Ser Asp Ala Ala Val Asp Thr Ser Ser Glu Ile Thr Thr Lys Asp Leu 1 5 10 15 Lys Glu Lys Lys Glu Val Val Glu Glu Ala Glu Asn 20 25

Claims

【Claim 1】 The invention described in the specification and drawings of this application.

Images