WO2016033187A1 - Use of gelsolin in the treatment and diagnosis of sickle cell disease - Google Patents

Abstract

The disclosure involves the administration of gelsolin to a subject to treat a Sickle Cell Disease (SCO) complication. The disclosure is also directed to methods of using gelsolin to diagnose or predict a SCD complication and to monitor the effect of treatment. According to one aspect, a method of treating a subject having or at risk of developing a SCD complication is provided. The method comprises administering to a subject in need of such a treatment an effective amount of gelsolin to treat the subject. According to another aspect of the disclosure, a method of treating a subject having or at risk of developing a SCD complication to reduce the risk of the SCD complication is provided. The method comprises administering to a subject in need of such a treatment an effective amount of gelsolin to raise the level of gelsolin in the subject above a reference level.

Description

USE OF GELSOLIN IN THE TREATMENT AND DIAGNOSIS OF SICKLE CELL

DISEASE

Related Application

This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Patent Application filed August 26, 2014, entitled "USE OF GELSOLIN IN THE

TREATMENT AND DIAGNOSIS OF SICKLE CELL DISEASE", Serial No. 62/042,196, the content of which is incorporated by reference in its entirety.

Field of the Invention

The disclosure is directed to therapeutic and diagnostic uses of gelsolin in sickle cell disease.

Background of the Invention

Red blood cell destruction (hemolysis) is a central complication of the genetic condition, Sickle Cell Disease (SCD) caused by a homozygous hemoglobin mutation.

Hemolysis in SCD is responsible for severe anemia and contributes to other SCD

complications. Despite advances in diagnosis and therapy, SCD remains a major cause of morbidity and mortality. There is a strong incentive to identify new treatments for SCD complications.

Because the outcome of treatment depends on a proper diagnosis, it is important to have proper tests to diagnose SCD and its complications and to monitor the treatment of the SCD complications. A precise diagnosis permits a physician to institute proper and timely therapy. Thus, there is also a strong incentive to identify new improved tests and approaches to diagnose and to evaluate treatments of SCD in addition to identifying treatment candidates.

Summary of the Invention

A unique feature of hemolysis in SCD is that the major site of red blood cell destruction is in small blood vessels in the venous circulation. There, low oxygen tension promotes polymerization of the mutant hemoglobin leading to deformation and rupture of the red blood cell membrane (sickling). The misshapen red cells aggregate and block blood flow (Buchanan et al, Severe sickle cell disease-pathophysiology and therapy, Biel Blood Marrow Transplant 16 (Suppl 1): S64- S66, 2010). Compounding this stasis is release from the damaged red cells of lipid mediators that recruit blood platelets and white blood cells to the sickling site (Kuypers, Hemoglobin S polymerization and red blood cell membrane changes. Hematol Oncol Clin North Am 28: 155-179, 2014), further compromising fluid flow. The circulatory impairment worsens local hypoxia, thereby enhancing the sickling of red blood cells.

The red blood cell membrane cytoskeleton contains actin, and membrane damage accompanying hemolysis results in sequestration of the actin-binding protein plasma gelsolin (pGSN) (Smith et al, Decreased gelsolin levels in patients with Plasmodium falciparum malaria - a consequence of hemolysis? Blood 72: 214-218, 1988). The Applicant believes that gelsolin (e.g., pGSN) depletion in SCD contributes to the cycle of events described above that cause hemolysis and that pGSN repletion will therefore ameliorate the hemolytic cycle.

Without wishing to be bound by any particular mechanism or theory, it is believed that the mechanism to account for such amelioration include gelsolin' s (e.g., pGSN) inactivation of the mediators that compound capillary flow restriction through

vasoconstriction and recruitment of platelets and white blood cells (Osborn et al, Am J Physiol Cell Physiol 292: 1323-1330, 2007). These effects, amplified by repletion of gelsolin (e.g., pGSN) in SCD patients, are predicted to reduce hemolysis intensity, a parameter measureable by reduced signs of hemolysis that are consistently abnormal in SCD: anemia, high reticulocyte count, and elevated serum bilirubin and LDH.

Thus, the disclosure involves, in one aspect, the administration of gelsolin (e.g., pGSN) to a subject to treat a SCD complication. The disclosure is also directed to methods of using gelsolin (e.g., pGSN) to diagnose or predict a SCD complication and to monitor the effect of treatment.

According to one aspect of the disclosure, a method of treating a subject having or at risk of developing a SCD complication is provided. The method comprises administering to a subject in need of such a treatment an effective amount of gelsolin to treat the subject.

According to another aspect of the disclosure, a method of treating a subject having or at risk of developing a SCD complication to reduce the risk of the SCD complication is provided. The method comprises administering to a subject in need of such a treatment an effective amount of gelsolin to raise the level of gelsolin in the subject above a reference level.

In some embodiments, the subject is otherwise free of indications calling for treatment with gelsolin. In some embodiments, the gelsolin is plasma gelsolin (pGSN) or cytoplasmic gelsolin (cGSN). The gelsolin may be administered orally, sublingually, buccally, intranasally, intravenously, intramuscularly, intrathecally, intraperitoneally, subcutaneously, or by inhalation. In some embodiments, the gelsolin is administered prophylactically.

In some embodiments, the method further comprises administering a second agent for treating a SCD complication. In some embodiments, the second agent is oxygen inhalation, hydroxyurea, folic acid, an iron chelator, heparin, pentosan polysulfate, diphenhydramine, nitric oxide, L-arginine, eptifibatide, prasugrel, zileuton (Zyflo CR), a statin, a corticosteroid, a glucocorticoid, regadenoson (Lexiscan™), 5-hydroxymethyl-2-furfural (Aes-103), a selectin antagonist, a selectin antibody, humanized antibody SelGl, Rivipansel, an integrin antagonist, an integrin antibody, an analgesic, an anti-inflammatory agent, oxygen inhalation, intravenous hydration, or a blood transfusion.

In some embodiments, the subject undergoes an exchange transfusion.

According to another aspect of the disclosure, a method of treating a subject at an increased risk of developing a SCD complication to reduce the risk of a SCD complication is provided. The method comprises selecting a subject on the basis that the subject is known to have a level of gelsolin below a reference level and administering to the subject gelsolin and/or a second agent in an effective amount to reduce the subject's risk of developing a SCD complication.

According to another aspect of the disclosure, a method of treating a subject at an increased risk of developing a SCD complication to reduce the risk of a SCD complication is provided. The method comprises selecting the subject on the basis that the subject is known to have a level of gelsolin below a reference level and administering to the subject gelsolin and/or a second agent in an effective amount to raise the level of gelsolin in the subject above the reference level.

In some embodiments, the subject is otherwise free of indications calling for treatment with gelsolin. In some embodiments, the subject is apparently healthy.

In some embodiments, the gelsolin is plasma gelsolin (pGSN) or cytoplasmic gelsolin (cGSN).

In some embodiments, the method further comprises administering a second agent for treating a SCD complication. In some embodiments, the second agent is hydroxyurea, folic acid, an iron chelator, heparin, pentosan polysulfate, diphenhydramine, nitric oxide, L- arginine, eptifibatide, prasugrel, zileuton (Zyflo CR), a statin, a corticosteroid, a

glucocorticoid, regadenoson (Lexiscan™), 5-hydroxymethyl-2-furfural (Aes-103), a selectin antagonist, a selectin antibody, humanized antibody SelGl, Rivipansel, an integrin antagonist, an integrin antibody, an analgesic, an anti-inflammatory agent, oxygen inhalation, intravenous hydration, or a blood transfusion.

According to another aspect, the disclosure provides gelsolin for use in a method for treating a subject having or at risk of developing a SCD complication.

In some embodiments, the subject is otherwise free of indications calling for treatment with gelsolin.

In some embodiments, the gelsolin is plasma gelsolin (pGSN) or cytoplasmic gelsolin (cGSN). In some embodiments, the gelsolin is for administration orally, sublingually, buccally, intranasally, intravenously, intramuscularly, intrathecally, intraperitoneally, subcutaneously, or by inhalation.

In some embodiments, the gelsolin is for use with a second agent wherein the second agent is hydroxyurea, folic acid, an iron chelator, heparin, pentosan polysulfate,

diphenhydramine, nitric oxide, L-arginine, eptifibatide, prasugrel, zileuton (Zyflo CR), a statin, a corticosteroid, a glucocorticoid, regadenoson (Lexiscan™), 5-hydroxymethyl-2- furfural (Aes-103), a selectin antagonist, a selectin antibody, humanized antibody SelGl, Rivipansel, an integrin antagonist, an integrin antibody, an analgesic, an anti-inflammatory agent, oxygen inhalation, intravenous hydration, or a blood transfusion.

According to another aspect, the disclosure provides gelsolin for use in a method for treating a subject having or at risk of developing a SCD complication wherein the subject is selected on the basis that the subject is known to have a level of gelsolin below a reference level.

In some embodiments, the subject is otherwise free of indications calling for treatment with gelsolin.

In some embodiments, the gelsolin is plasma gelsolin (pGSN) or cytoplasmic gelsolin (cGSN). In some embodiments, the gelsolin is for administration orally, sublingually, buccally, intranasally, intravenously, intramuscularly, intrathecally, intraperitoneally, subcutaneously, or by inhalation.

In some embodiments, the gelsolin is for use with a second agent wherein the second agent is hydroxyurea, folic acid, an iron chelator, heparin, pentosan polysulfate,

diphenhydramine, nitric oxide, L-arginine, eptifibatide, prasugrel, zileuton (Zyflo CR), a statin, a corticosteroid, a glucocorticoid, regadenoson (Lexiscan™), 5-hydroxymethyl-2- furfural (Aes-103), a selectin antagonist, a selectin antibody, humanized antibody SelGl, Rivipansel, an integrin antagonist, an integrin antibody, an analgesic, an anti-inflammatory agent, oxygen, intravenous hydration, or a blood transfusion.

According to yet another aspect, the disclosure provides gelsolin in the manufacture of a medicament for treating a subject having or at risk of developing a SCD complication.

In some embodiments, the subject is otherwise free of indications calling for treatment with gelsolin.

In some embodiments, the gelsolin is plasma gelsolin (pGSN) or cytoplasmic gelsolin (cGSN). In some embodiments, the gelsolin is for administration orally, sublingually, buccally, intranasally, intravenously, intramuscularly, intrathecally, intraperitoneally, subcutaneously, or by inhalation.

In some embodiments, the medicament comprises a second agent wherein the second agent is hydroxyurea, folic acid, an iron chelator, heparin, pentosan polysulfate,

diphenhydramine, nitric oxide, L-arginine, eptifibatide, prasugrel, zileuton (Zyflo CR), a statin, a corticosteroid, a glucocorticoid, regadenoson (Lexiscan™), 5-hydroxymethyl-2- furfural (Aes-103), a selectin antagonist, a selectin antibody, humanized antibody SelGl, Rivipansel, an integrin antagonist, an integrin antibody, an analgesic, or an anti-inflammatory agent.

According to still another aspect, the disclosure provides gelsolin in the manufacture of a medicament for treating a subject having or at risk of developing a SCD complication wherein the subject on the basis that the subject is known to have a level of gelsolin below a reference level.

In some embodiments, the subject is otherwise free of indications calling for treatment with gelsolin.

In some embodiments, the gelsolin is plasma gelsolin (pGSN) or cytoplasmic gelsolin (cGSN). In some embodiments, the gelsolin is for administration orally, sublingually, buccally, intranasally, intravenously, intramuscularly, intrathecally, intraperitoneally, subcutaneously, or by inhalation.

In some embodiments, the medicament comprises a second agent wherein the second agent is hydroxyurea, folic acid, an iron chelator, heparin, pentosan polysulfate,

diphenhydramine, nitric oxide, L-arginine, eptifibatide, prasugrel, zileuton (Zyflo CR), a statin, a corticosteroid, a glucocorticoid, regadenoson (Lexiscan™), 5-hydroxymethyl-2- furfural (Aes-103), a selectin antagonist, a selectin antibody, humanized antibody SelGl, Rivipansel, an integrin antagonist, an integrin antibody, an analgesic, or an anti-inflammatory agent. According to yet another aspect of the disclosure, a method of characterizing a subject's risk of developing a SCD complication is provided. The method comprises comparing a level of gelsolin in a sample from the subject to a reference level, and characterizing the subject's risk of developing a SCD complication based upon the level of gelsolin in comparison to the reference level, wherein a level of gelsolin below the reference level is indicative that the subject is at an elevated risk of developing a SCD complication, and/or wherein a level of gelsolin at or above the reference level is indicative that the subject is not at an elevated risk of developing a SCD complication.

In some embodiments, the method further comprises determining the level of gelsolin in the sample. In some embodiments, the method further comprises providing a report indicating that the subject is at an elevated risk of developing a SCD complication when the level of gelsolin is below the reference level. In some embodiments, the method further comprises providing a report indicating that the subject is not at an elevated risk of developing a SCD complication when the level of gelsolin is at or above the reference level.

In some embodiments, the gelsolin is plasma gelsolin (pGSN) or cytoplasmic gelsolin (cGSN). In some embodiments, the level of gelsolin is in a body fluid of the subject. In some embodiments, the body fluid is blood, plasma, serum, urine, or saliva.

In some embodiments, the reference level is about 250 mg/L of plasma. In some embodiments, the reference level is 250 mg/L of plasma.

In some embodiments, the subject is an apparently healthy subject.

In some embodiments, the method further comprises performing one or more tests to evaluate SCD or a complication thereof. The test may be a blood reticulocyte count, blood smear inspection, hemoglobin level, hemoglobin electrophoresis, hemoglobin S, or sickle cell index.

According to still another aspect of the disclosure, a method of evaluating the efficacy of a treatment of a SCD complication in a subject is provided. The method comprises (i) determining a level of gelsolin in a sample from the subject undergoing treatment with an agent to treat or reduce the risk of a SCD complication, (ii) comparing the level of gelsolin obtained in (i) to a reference level corresponding to a level of gelsolin in an apparently healthy control population, and (iii) determining whether the level of gelsolin in (i) is at or above the reference level, said determination being indicative that the treatment is efficacious.

In some embodiments, the step (i) and step (ii) and /or step (iii) are repeated so as to monitor the level(s) of gelsolin over time. In some embodiments, the reference level is about 250 mg/L of plasma. In some embodiments, the reference level is 250 mg/L of plasma.

In some embodiments, the agent is gelsolin, hydroxyurea, folic acid, an iron chelator, heparin, pentosan polysulfate, diphenhydramine, nitric oxide, L-arginine, eptifibatide, prasugrel, zileuton (Zyflo CR), a statin, a corticosteroid, a glucocorticoid, regadenoson (Lexiscan™), 5-hydroxymethyl-2-furfural (Aes-103), a selectin antagonist, a selectin antibody, humanized antibody SelGl, Rivipansel, an integrin antagonist, an integrin antibody, an analgesic, an anti-inflammatory agent, oxygen inhalation, intravenous hydration, or a blood transfusion.

According to still another aspect of the disclosure, a method of deciding on the course of a treatment in a subject is provided. The method comprises (i) determining a level of gelsolin in a sample from the subject undergoing a treatment to treat or reduce the risk of a SCD complication, (ii) comparing the level of gelsolin obtained in (i) to a reference level corresponding to a level of gelsolin corresponding to a level of gelsolin in an apparently healthy control population, (iii) determining whether the level of gelsolin obtained in (i) is at or below the reference level, and (iv) deciding on the course of the treatment based on such determination.

In some embodiments, the step (i) and step (ii) and /or step (iii) are repeated so as to monitor the level(s) of gelsolin over time.

In some embodiments, the reference level is about 250 mg/L of plasma or lower.

A level of gelsolin below the reference level is a gelsolin level that is at least 10% less than the measured mean level for a given population of subjects. The mean level of gelsolin can depend upon the particular population of subjects. For example, an apparently healthy population will have a different "normal" range of gelsolin than will a population of subjects which have had a prior condition. In some embodiments, the level of gelsolin is at least 10% less than the measured mean level for a given population of subjects. In some embodiments, the level of gelsolin is at least 20% less than the measured mean level for a given population of subjects. In some embodiments, the level of gelsolin is at least 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 99% or 100% less than the measured mean level for a given population of subjects. In some embodiments, the level of gelsolin is below about 250 mg/L of plasma. In some embodiments, the level of gelsolin is below about 2.4 μΜ/L

(micromoles/Liter) of plasma. In other embodiments, the subject is apparently healthy. As used herein an "apparently healthy subject" is a subject who has no signs and/or symptoms of a disease.

In some embodiments, the reference level is 250 mg/L of plasma or lower. In some embodiments, the reference level of gelsolin is about 240 mg/L, 230 mg/L, 220 mg/L, 210 mg/L, 200 mg/L, 190 mg/L, 180 mg/L, 170 mg/L, 160 mg/L, 150 mg/L, 140 mg/L, 130 mg/L, 120 mg/L, 110 mg/L, 100 mg/L, 90 mg/L, 80 mg/L, 70 mg/L, 60 mg/L, 50 mg/L, 40 mg/L, 30 mg/L, 20 mg/L, or 10 mg/L of plasma or lower.

In some other embodiments, the reference level is 2.4 μΜ/L of plasma or lower. In some embodiments, the reference level of gelsolin is about 2.3 μΜ/L, 2.2 μΜ/L, 2.1 μΜ/L, 2.0 μΜ/L, 1.9 μΜ/L, 1.8 μΜ/L, 1.7 μΜ/L, 1.6 μΜ/L, 1.5 μΜ/L, 1.4 μΜ/L, 1.3 μΜ/L, 1.2 μΜ/L, 1.1 μΜ/L, 1.0 μΜ/L, 0.9 μΜ/L, 0.8 μΜ/L, 0.7 μΜ/L, 0.6 μΜ/L, 0.5 μΜ/L, 0.4 μΜ/L, 0.3 μΜ/L, 0.2 μΜ/L of plasma or lower.

In some embodiments the subject is otherwise free of indications calling for treatment with the agent. When the agent is gelsolin, a subject free of indications calling for treatment with gelsolin is a subject who has no signs or symptoms calling for treatment with gelsolin. Gelsolin is indicated for the treatment of sepsis, infections, multiple sclerosis, and rheumatoid arthritis. Gelsolin is indicated for the treatment of actin-related disorders such as Adult Respiratory Distress Syndrome (ARDS), fulminant hepatic necrosis, acute renal failure, muscle injury, disorders characterized by elevated levels of BUN and/or creatinine. Actin- related disorders are known to those of ordinary skill in the art.

Each of the limitations of the disclosure can encompass various embodiments of the disclosure. It is, therefore, anticipated that each of the limitations of the disclosure involving any one element or combinations of elements can be included in each aspect of the disclosure. The disclosure is capable of other embodiments and of being practiced or of being carried out in various ways. Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including", "comprising", or "having", "containing", "involving", and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

These and other aspects of the disclosure, as well as various advantages and utilities will be apparent with reference to the Detailed Description of the Invention. Each aspect of the disclosure can encompass various embodiments as will be understood.

All documents identified in this application are incorporated in their entirety herein by reference. Brief Description of the Drawings

FIG. 1 is the standard curve for rhu-pGSN as determined for sample assessment.

Detailed Description of the Invention

The instant disclosure involves, in some aspects, administering gelsolin to a subject for the treatment of SCD in the subject. The term "treatment" or "treating" a SCD

complication (one or more) is intended to include prophylaxis, amelioration, or prevention of a SCD complication (one or more), or reduction in the risk of developing a SCD complication (one or more). Treatment or treating a SCD complication (one or more) encompasses treatment of a manifestation (one or more) and/or a sign (one or more) and/or a symptom (one or more) of a SCD complication. SCD complications are known to those of ordinary skill in the art. A SCD complication can be an acute complication, a chronic complication or a combination thereof. Examples of acute SCD complications include, but are not limited to, pain crisis, acute chest, infection susceptibility, strokes, bone necrosis, avascular hip necrosis, and priapism. Examples of chronic SCD complications include, but are not limited to, hemolysis and its complications such as gall stones, anemia, renal deterioration, pulmonary hypertension, endocrine dysfunction, and Moyamoya.

As used herein the term "subject" means any mammal that may be in need of treatment. Preferred subjects are human subjects.

As used herein the term "gelsolin" encompasses wild type gelsolin (GenBank accession No.: X04412), isoforms, analogs, variants, fragments or functional derivatives of gelsolin.

Gelsolin, unlike other mammalian proteins, has both cytoplasmic (cGSN) and secreted or exported isoforms, also called plasma gelsolin (pGSN), which are derived by alternative splicing of the message from a single gene (Sun et al. J. Biol. Chem. 274:33179- 33182 (1999)). As used herein, gelsolin isoforms include versions of gelsolin with some small differences in their amino acid sequences, usually a splice variant or the result of some posttranslational modification.

Gelsolin encompasses native as well as synthetic and recombinant gelsolin and gelsolin analogs. Gelsolin is an abundant secretory protein (Yin, H. L., Kwiatkowski, D. J., Mole, J. E. & Cole, F. S. (1984) J Biol Chem 259, 5271-6). The exported isoform of gelsolin, pGSN, has 25 additional amino acids and originates from alternative splicing of a single gene (Kwiatkowski, D. J., Stossel, T. P., Orkin, S. H., Mole, J. E., Colten, H. R. & Yin, H. L. (1986) Nature 323, 455-8). Recombinant human gelsolin (rhGSN) (Biogen IDEC, Inc., Cambridge, MA) is produced in E. coli, and though it has the same primary structure as the native protein, under standard conditions of purification, it differs from natural human plasma gelsolin by a disulfide bond that is present in the natural protein. The recombinant protein is, therefore, properly oxidized after purification, and its structure and functions are

indistinguishable from human plasma gelsolin (Wen et. al., Biochemistry 35:9700-9709 (1996)). In some therapeutic aspects and embodiments of the disclosure, the use of rhGSN is preferred. In some embodiments, pGSN is preferred.

A "gelsolin analog" refers to a compound substantially similar in function to either the native gelsolin or to a fragment thereof. Gelsolin analogs include biologically active amino acid sequences substantially similar to the gelsolin sequences and may have substituted, deleted, elongated, replaced, or otherwise modified sequences that possess bioactivity substantially similar to that of gelsolin. For example, an analog of gelsolin is one which does not have the same amino acid sequence as gelsolin but which is sufficiently homologous to gelsolin so as to retain the bioactivity of gelsolin. Bioactivity can be determined, for example, by determining the properties of the gelsolin analog and/or by determining the ability of the gelsolin analog to treat or prevent SCD. One example of a gelsolin bioactivity assay is gelsolin' s ability to stimulate actin nucleation. Gelsolin bioactivities are known to those of ordinary skill in the art.

A "fragment" is meant to include any portion of a gelsolin molecule which provides a segment of gelsolin which maintains the bioactivity of gelsolin; the term is meant to include gelsolin fragments which are made from any source, such as, for example, from naturally- occurring peptide sequences, synthetic or chemically- synthesized peptide sequences, and genetically engineered peptide sequences.

A "variant" of gelsolin is meant to refer to a compound substantially similar in structure and bioactivity either to native gelsolin, or to a fragment thereof. The term variant encompasses the gelsolin family of proteins. The gelsolin family of proteins is a group of actin binding proteins sharing repeats of about 15kDa homologous domains that adopt a similar fold. Examples gelsolin family proteins include but are not limited to advillin, villin, capG, flightless proteins, fragmin, severin, adseverin, protovillin, and supervillin.

A "functional derivative" of gelsolin is a derivative which possesses a bioactivity that is substantially similar to the bioactivity of gelsolin. By "substantially similar" is meant activity which is quantitatively different but qualitatively the same. For example, a functional derivative of gelsolin could contain the same amino acid backbone as gelsolin but also contains other modifications such as post-translational modifications such as, for example, bound phospholipids, or covalently linked carbohydrate, depending on the necessity of such modifications for the performance of the diagnostic assay or therapeutic treatment. As used herein, the term is also meant to include a chemical derivative of gelsolin. Such derivatives may improve gelsolin's solubility, absorption, biological half-life, etc. The derivatives may also decrease the toxicity of gelsolin, or eliminate or attenuate any undesirable side effect of gelsolin, etc. Chemical moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980). Procedures for coupling such moieties to a molecule such as gelsolin are well known in the art. The term "functional derivative" is intended to include the "fragments," "variants," "analogues," or "chemical derivatives" of gelsolin.

The disclosure involves in some aspects, methods for treating a SCD complication in a subject. The subject is known to have, is suspected of having, or is at risk of having a SCD complication. The gelsolin is administered in an amount effective to treat the SCD complication in the subject.

A response to a treatment method of the disclosure can, for example, be measured by determining the physiological effects of the treatment, such as the decrease or lack of a SCD complication following administration of the treatment.

In another aspect of the disclosure, a method for monitoring treatment in a subject is provided. The method involves obtaining a level of gelsolin in a subject undergoing treatment to treat a SCD complication. The level of gelsolin is compared to a reference level corresponding to a control level of gelsolin (e.g., in an apparently healthy population). A determination of whether the level of gelsolin is at or below a reference level is indicative of whether the subject would benefit from continued treatment with the same treatment or would benefit from a change in treatment. In some embodiments, obtaining a level of gelsolin is repeated so as to monitor the subject's levels of gelsolin over time. In some embodiments, the subject may have been undergoing the treatment for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or more. In some embodiments, the subject may have been undergoing the treatment for at least 3, 4, 5, 6 months or more.

A change in treatment with gelsolin refers to an increase in the dose of the gelsolin, a switch from gelsolin to another agent, the addition of another agent to the gelsolin therapeutic regimen, or a combination thereof.

According to another aspect of the disclosure, a method for evaluating the efficacy of a treatment for a SCD complication is provided. The method involves obtaining a level of gelsolin in a subject undergoing treatment of the SCD complication. The level of gelsolin is compared to a reference level corresponding to a control level of gelsolin (e.g., in an apparently healthy population). A determination that the level of gelsolin is at or above a reference level would be indicative that the treatment is efficacious. In some embodiments, the subject may have been undergoing the treatment for at least at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more weeks. In some embodiments, the subject may have been undergoing the treatment for at least 3, 4, 5, 6, or more months.

One aspect of the disclosure is directed to the measurement of gelsolin to guide treatments in order to improve outcome in subjects. On-treatment levels of gelsolin have predictive value for response to a treatment(s) of a SCD complication(s). The on-treatment levels of gelsolin are additive to prior art predictors of outcome of the SCD complication(s).

Subjects who would benefit from this aspect of this disclosure are subjects who are undergoing treatment to treat or prevent a SCD complication. A subject on-treatment is a subject who already has been diagnosed and is in the course of treatment for a SCD complication. The treatment can be any of the therapeutic agents referred to herein. The treatment also can be non-drug treatments. In important embodiments, the treatment is one which increases levels of gelsolin. In a particularly important embodiment, the treatment is a treatment with gelsolin. Preferred subjects are human subjects. The subject most likely to benefit from this invention is a human subject on-treatment and who has a gelsolin level at or below about 250 mg/L (or 2.4 μΜ/L) of plasma.

In some embodiments, the subject already has a SCD disease complication. In some embodiments, the subject may be at an elevated risk of having a such complication.

Risk factors for diseases are known to those of ordinary skill in the art. For example, risk factors for SCD and a SCD complication include ethnicity and family history. At least five haplotypes of sickle cell disease are recognized based upon their origin: Senegal, Cameroon, Benin, Central African Republic, and India. Risk charts and prediction algorithms may be used for assessing the risk of a SCD complication(s) in a subject based on the presence and severity of risk factors. In some embodiments, the subject who is at an elevated risk of having a SCD complication(s) may be an apparently healthy subject. An apparently healthy subject is a subject who has no signs or symptoms of disease. Other methods of assessing the risk of a SCD complication(s) in a subject are known by those of ordinary skill in the art.

The preferred treatment of the instant disclosure is gelsolin. Gelsolin may be administered alone, in a pharmaceutical composition or combined with other therapeutic regimens. Gelsolin and optionally other treatment or therapeutic agent(s) may be administered simultaneously or sequentially. When the other treatment or therapeutic agents are administered simultaneously they can be administered in the same or separate

formulations, but are administered at the same time. The other treatment or therapeutic agents may be administered sequentially with one another and with gelsolin when the administration of the other treatment or therapeutic agents and the gelsolin is temporally separated. The separation in time between the administration of these agents may be a matter of minutes or it may be longer.

In practicing certain methods of the present disclosure, it is required to obtain a level of gelsolin in a subject. This level then is compared to a reference level, wherein the level of gelsolin in comparison to the reference level is indicative of the likelihood that the subject will benefit from continued treatment. The subject then can be characterized in terms of the net benefit likely to be obtained from a change in treatment.

The level of the gelsolin for the subject can be obtained by any art recognized method. Typically, the level is determined by measuring the level of gelsolin in a body fluid, for example, blood, serum, plasma, lymph, saliva, urine and the like. The level can be determined by ELISA, or other immunoassays or other conventional techniques for determining the presence of gelsolin. Conventional methods may include sending a sample(s) of a subject's body fluid to a commercial laboratory for measurement.

The disclosure also involves comparing the level of gelsolin for the subject with a reference level. The reference level can take a variety of forms. It can be single cut-off value, such as a median or mean. It can be established based upon comparative groups, such as, for example, where the risk in one defined group is double the risk in another defined group. It can be a range, for example, where the tested population is divided equally (or unequally) into groups, such as a low-risk group, a medium-risk group and a high-risk group, or into quartiles, the lowest quartile being subjects with the highest risk and the highest quartile being subjects with the lowest risk, or into tertiles the lowest tertile being subjects with the highest risk and the highest tertile being subjects with the lowest risk. The reference level may be a cut-off value which is predetermined by the fact that a group having a gelsolin level no less than the cut-off value demonstrates a statistically significant increase in the risk of developing a SCD complication as compared to a comparative group. In some

embodiments the comparative group is a group having a lower level of gelsolin.

The reference level can depend upon the particular population of subjects selected. For example, an apparently healthy population may have a different 'normal' range of gelsolin than will populations of subjects of which have other conditions. Accordingly, the reference levels selected may take into account the category in which a subject falls.

Appropriate ranges and categories can be selected with no more than routine experimentation by those of ordinary skill in the art.

The preferred body fluid is blood. In some embodiments, the reference level of gelsolin is about 250 mg/L of plasma or lower. In some embodiments, reference level of gelsolin is about 240 mg/L, 230 mg/L, 220 mg/L, 210 mg/L, 200 mg/L, 190 mg/L, 180 mg/L, 170 mg/L, 160 mg/L, 150 mg/L, 140 mg/L, 130 mg/L, 120 mg/L, 110 mg/L, 100 mg/L, 90 mg/L, 80 mg/L, 70 mg/L, 60 mg/L, 50 mg/L, 40 mg/L, 30 mg/L, 20 mg/L, or 10 mg/L of plasma or lower.

In some embodiments, the reference level of gelsolin is about 2.4 μΜ/L of plasma or lower. In some embodiments, the reference level of gelsolin is about 2.3 μΜ/L, 2.2 μΜ/L, 2.1 μΜ/L, 2.0 μΜ/L, 1.9 μΜ/L, 1.8 μΜ/L, 1.7 μΜ/L, 1.6 μΜ/L, 1.5 μΜ/L, 1.4 μΜ/L, 1.3 μΜ/L, 1.2 μΜ/L, 1.1 μΜ/L, 1.0 μΜ/L, 0.9 μΜ/L, 0.8 μΜ/L, 0.7 μΜ/L, 0.6 μΜ/L, 0.5 μΜ/L, 0.4 μΜ/L, 0.3 μΜ/L, 0.2 μΜ/L of plasma or lower.

In some embodiments, the reference level of gelsolin is a value that is the average for a healthy subject population (i.e., subjects who have no signs and symptoms of disease). The reference level will depend, of course, upon the characteristics of the subject population in which the subject lies. In characterizing risk, numerous reference levels can be established. Presently, there are commercial sources which produce reagents for assays for gelsolin.

These include, for example, Cytoskeleton (Denver, CO), Sigma (St. Louis, MO) and

Calbiochem (San Diego, CA)

In some embodiments, the disclosure further comprises measuring the level of gelsolin together with a level of a second test for SCD. Examples of tests for SCD include, for example, blood reticulocyte count, blood smear inspection (e.g., for sickled red blood cells), hemoglobin level, hemoglobin electrophoresis, hemoglobin S, and sickle cell index

In some embodiments, a level of gelsolin in the subject is obtained. The level of gelsolin is compared to a reference level to establish a first risk value. A level of the second marker in the subject is also obtained. The level of the second marker in the subject is compared to a second reference level to establish a second risk value. The subject's risk profile of developing the disease then is characterized based upon the combination of the first risk value and the second risk value, wherein the combination of the first risk value and second risk value establishes a third risk value different from the first and second risk values. In some embodiments, the third risk value is greater than either of the first and second risk values. The preferred subjects for testing and reference levels are as described above. The disclosure provides methods for determining whether a subject will benefit from continued treatment or would benefit from a change in treatment. The benefit is typically a reduction in the signs and symptoms or a faster recovery from the complication(s) of SCD. Complications of SCD are known to those of ordinary skill in the art.

SCD complications are heterogeneous. Some patients remain virtually asymptomatic into or even through adult life, while others suffer repeated crises requiring hospitalization from early childhood. Most patients with SCD suffer from hemolytic anemia, with hematocrits of 15 to 30%, and significant reticulocytosis. Granulocytosis is common. The white blood cell count can fluctuate substantially and unpredictably during and between painful crises, infectious episodes, and other intercurrent illnesses.

Vasoocclusion in SCD causes protean complications; intermittent episodes in connective and musculoskeletal structures produce painful ischemia manifested by acute pain and tenderness, fever, tachycardia, and anxiety. These recurrent episodes, known as painful crises, are the most common clinical manifestation. Their frequency and severity vary greatly. Pain can develop almost anywhere in the body and may last from a few hours to 2 weeks. Repeated crises requiring hospitalization (more than three per year) correlate with reduced survival in adult life, suggesting that these episodes are associated with accumulation of chronic end-organ damage. Provocative factors include infection, fever, excessive exercise, anxiety, abrupt changes in temperature, hypoxia, or hypertonic dyes.

Repeated microinfarction can destroy tissues having microvascular beds that promote sickling. Thus, the spleen is frequently infarcted within the first 18 to 36 months of life, causing susceptibility to infection, particularly from pneumococci. Occlusion of retinal vessels can produce hemorrhage, neovascularization, and eventual detachments. More widespread renal necrosis leads to renal failure in adults. Bone and joint ischemia can lead to aseptic necrosis (especially of the femoral or humeral heads), chronic arthropathy, and unusual susceptibility to osteomyelitis which may be caused by organisms such as

Salmonella, rarely encountered in other settings. The hand-foot syndrome is caused by painful infarcts of the digits and dactylitis. Stroke is especially common in children, a small subset of whom tend to suffer repeated episodes; stroke is less common in adults and is often hemorrhagic. Chronic lower leg ulcers probably arise from ischemia and superinfection in the distal circulation.

Acute chest syndrome is a distinctive complication characterized by chest pain, tachypnea, fever, cough, and arterial oxygen desaturation. It can mimic pneumonia, pulmonary emboli, bone marrow infarction and embolism, myocardial ischemia or in situ lung infarction. Acute chest syndrome is thought to reflect in situ sickling within the lung, producing pain and temporary pulmonary dysfunction. Acute chest syndrome may be difficult or impossible to distinguish from other entities. Pulmonary infarction and pneumonia are frequent underlying or concomitant conditions in patients with SCD.

Repeated episodes of acute chest pain correlate with reduced survival. Acutely, reduction in arterial oxygen saturation is especially ominous because it promotes sickling on a large scale. Repeated acute or subacute pulmonary crises lead to pulmonary hypertension and cor pulmonale.

The methods described herein have important implications for patient treatment and also for the clinical development of new treatments. Determining whether a subject will benefit from continued therapy or would benefit from a change in therapy is clinically useful. One example of clinical usefulness of the methods of this disclosure includes identifying subjects who are less likely or more likely to respond to a treatment. The methods of the disclosure are also useful in predicting or determining that a subject would benefit from continued treatment or would benefit from a change in treatment. Health care practitioners select therapeutic regimens for treatment based upon the expected net benefit to the subject. The net benefit is derived from the risk to benefit ratio. The present disclosure permits the determination of whether a subject will benefit from continued treatment or would benefit from a change in treatment, thereby aiding the physician in selecting a treatment.

Another example of clinical usefulness, in the case of human subjects for example, includes aiding clinical investigators in the selection for clinical trials of subjects with a high likelihood of obtaining a net benefit. It is expected that clinical investigators now will use the present disclosure for determining entry criteria for clinical trials.

A subject who would benefit from continued treatment is a subject whose on- treatment level of gelsolin reaches a certain reference level or whose level of gelsolin is increasing. Reference levels of gelsolin are described above. A subject who would benefit from a change in treatment is a subject whose on-treatment level of the gelsolin did not reach a certain reference level or whose on-treatment level of gelsolin is not increasing.

As used herein, a "change in treatment" refers to an increase or decrease in the dose of the existing treatment, a switch from one treatment to another treatment, an addition of another treatment to the existing treatment, or a combination thereof. A switch from one treatment to another may involve a switch to a treatment with a high risk profile but where the likelihood of expected benefit is increased. In some embodiments, preferred treatments are treatments that increase the levels of gelsolin. A subject who would benefit from a change in treatment by increasing the dose of the existing treatment is a subject who, for example, was on the treatment but was not receiving the maximum tolerated dose or the maximum allowed dose of the treatment and whose level of gelsolin did not reach a certain reference level. In such instances the dose of the existing treatment is increased until the level of gelsolin reaches a certain reference level. In some instances, the dose of the existing treatment is increased from the existing dose to a higher dose that is not the maximum tolerated dose nor the maximum allowed dose of the treatment. In other instances, the dose is increased to the maximum tolerated or to the maximum allowed dose of the treatment. A subject who would benefit from a change in treatment by decreasing the dose of the existing treatment is, for example, a subject whose on-treatment level of gelsolin reaches or can reach a certain reference level with a lower dose of the treatment.

A subject who would benefit from a switch from one treatment to another treatment is, for example, a subject who was on the maximum tolerated dose or the maximum allowed dose of the treatment and whose level of gelsolin did not reach a certain reference level. Another example is a subject was not on the maximum tolerated or the maximum allowed dose of the treatment but was determined by a health care practitioner to more likely benefit from another treatment. Such determinations are based, for example, on the development in the subject of unwanted side effects on the initial treatment or a lack of response to the initial treatment.

A subject who would benefit from a change in treatment by the addition of another treatment to the existing treatment is, for example, a subject who was on a treatment but whose level of gelsolin did not reach a certain reference level. In such instances, another treatment is added to the existing treatment. The treatment that is added to the existing treatment can have a different mechanism of action in increasing the level of gelsolin than the existing treatment. In some instances, a combination of the aforementioned changes in treatment may be used.

The disclosure also provides methods for determining the efficacy of a treatment. The efficacy is typically the efficacy of the treatment in increasing the level of gelsolin and/or treating a SCD complication. This is sometimes also referred to as a positive response or a favorable response. Efficacy can be determined by a gelsolin blood test(s) to determine whether gelsolin levels are increased as a result of treatment. In some embodiments efficacy determination is based on the efficacy of a treatment in increasing gelsolin and normalizing SCD tests (e.g., normalizing reticulocyte counts, levels of hemoglobin S, or sickle cell index) The gelsolin measurement typically is reported in μΜ/L (micromoles/Liter), mg/dl (milligrams/deciliter), or mg/L (milligrams/Liter).

The disclosure also provides methods for deciding on the course of a treatment in a subject undergoing treatment for a SCD complication. Such a course of treatment is decided on the basis of the level of gelsolin. In some embodiments, the subject has a SCD

complication. In some embodiments, the subject already has SCD or is at risk of having the disease. In some embodiments, the subject is at an elevated risk of having the disease the subject has one or more risk factors to have the disease.

The amount of a treatment may be varied for example by increasing or decreasing the amount of gelsolin or pharmacological agent or a treatment or therapeutic composition, by changing the treatment or therapeutic composition administered, by changing the route of administration, by changing the dosage timing and so on. The effective amount will vary with the particular condition being treated, the age and physical condition of the subject being treated, the severity of the condition, the duration of the treatment, the nature of the concurrent treatment (if any), the specific route of administration, and like factors are within the knowledge and expertise of the health practitioner. For example, an effective amount can depend upon the duration the individual has had SCD or a SCD complication.

An effective amount is a dosage of the therapeutic agent sufficient to provide a medically desirable result. An effective amount may also, for example, depend upon the degree to which an individual has abnormally decreased levels of gelsolin. It should be understood that the therapeutic agents of the disclosure are used to treat or prevent a SCD complication(s), that is, they may be used prophylactically in subjects at risk of developing a SCD complication(s). Thus, an effective amount is that amount which can lower the risk of, slow or perhaps prevent altogether the development of a SCD complication(s). It will be recognized when the therapeutic agent is used in acute circumstances, it is used to prevent one or more medically undesirable results that typically flow from such adverse events.

The factors involved in determining an effective amount are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is generally preferred that a maximum dose of the pharmacological agents of the disclosure (alone or in combination with other therapeutic agents) be used, that is, the highest safe dose according to sound medical judgment. It will be understood by those of ordinary skill in the art however, that a patient may insist upon a lower dose or tolerable dose for medical reasons, psychological reasons or for virtually any other reasons. The therapeutically effective amount of a therapeutic agent of the disclosure is that amount effective to treat the disease. For example, in the case of SCD, the desired response is preventing, delaying the onset of a SCD complication(s) for example, reducing or eliminating the signs and symptoms of SCD complications. This can be monitored by routine diagnostic methods known to those of ordinary skill in the art.

The therapeutic agents used in the methods of the disclosure are preferably sterile and contain an effective amount of gelsolin for producing the desired response in a unit of weight or volume suitable for administration to a subject. The doses of therapeutic agents administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject. Other factors include the desired period of treatment. In the event that a response in a subject is insufficient at the initial doses applied, higher doses (or effectively higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits. The dosage of a treatment or therapeutic agent may be adjusted by the individual physician. A therapeutically effective amount typically varies from 0.01 mg/kg to about 1000 mg/kg, preferably from about 0.1 mg/kg to about 500 mg/kg, and most preferably from about 0.2 mg/kg to about 250 mg/kg, in one or more dose administrations daily, for one or more days.

Various modes of administration are known to those of ordinary skill in the art which effectively deliver the therapeutic agents of the disclosure to a desired tissue, cell, or bodily fluid. The disclosure is not limited by the particular modes of administration disclosed herein. Standard references in the art (e.g., Remington's Pharmaceutical Sciences, 20th Edition, Lippincott, Williams and Wilkins, Baltimore MD, 2001) provide modes of administration and formulations for delivery of various pharmaceutical preparations and formulations in pharmaceutical carriers. Other protocols which are useful for the

administration of pharmacological agents of the disclosure will be known to one of ordinary skill in the art, in which the dose amount, schedule of administration, sites of administration, mode of administration and the like vary from those presented herein.

Administration of pharmacological agents of the disclosure to mammals other than humans, e.g. for testing purposes, is carried out under substantially the same conditions as described above. It will be understood by one of ordinary skill in the art that this disclosure is applicable to both human and animal diseases.

When administered, the pharmaceutical preparations of the disclosure are applied in pharmaceutically-acceptable amounts and in pharmaceutically- acceptable compositions. The term "pharmaceutically acceptable" means a non-toxic material that does not interfere with the effectiveness of the biological activity of the active ingredients. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents. When used in medicine, the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the disclosure. Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.

A pharmacological agent or composition may be combined, if desired, with a pharmaceutically-acceptable carrier. The term "pharmaceutically-acceptable carrier" as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances which are suitable for administration into a human. The term "carrier" denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being co-mingled with the pharmacological agents of the disclosure, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy.

The pharmaceutical compositions may contain suitable buffering agents, as described above, including: acetate, phosphate, citrate, glycine, borate, carbonate, bicarbonate, hydroxide (and other bases) and pharmaceutically acceptable salts of the foregoing compounds. The pharmaceutical compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.

The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier, which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

The treatments or therapeutic agents, when it is desirable to deliver them systemically, may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active compounds may be in powder form for constitution with a suitable vehicle (e.g., saline, buffer, or sterile pyrogen-free water) before use.

Compositions suitable for oral administration may be presented as discrete units, such as capsules, tablets, pills, lozenges, each containing a predetermined amount of the active compound (e.g., gelsolin). Other compositions include suspensions in aqueous liquids or non-aqueous liquids such as a syrup, elixir, an emulsion, or a gel.

Pharmaceutical preparations for oral use can be obtained as solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, sorbitol or cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl cellulose, sodium

carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Optionally the oral formulations may also be formulated in saline or buffers, i.e. EDTA for neutralizing internal acid conditions or may be administered without any carriers.

Also specifically contemplated are oral dosage forms of the above component or components. The component or components may be chemically modified so that oral delivery of the derivative is efficacious. Generally, the chemical modification contemplated is the attachment of at least one moiety to the component molecule itself, where said moiety permits (a) inhibition of proteolysis; and (b) uptake into the blood stream from the stomach or intestine. Also desired is the increase in overall stability of the component or components and increase in circulation time in the body. Examples of such moieties include:

polyethylene glycol, copolymers of ethylene glycol and propylene glycol, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinyl pyrrolidone and polyproline. Abuchowski and Davis, 1981, "Soluble Polymer-Enzyme Adducts" In: Enzymes as Drugs, Hocenberg and Roberts, eds., Wiley-Interscience, New York, NY, pp. 367-383; Newmark, et al., 1982, J. Appl. Biochem. 4: 185-189. Other polymers that could be used are poly-l,3-dioxolane and poly-l,3,6-tioxocane. Preferred for pharmaceutical usage, as indicated above, are

polyethylene glycol moieties.

For the component (or derivative) the location of release may be the stomach, the small intestine (the duodenum, the jejunum, or the ileum), or the large intestine. One skilled in the art has available formulations which will not dissolve in the stomach, yet will release the material in the duodenum or elsewhere in the intestine. Preferably, the release will avoid the deleterious effects of the stomach environment, either by protection of gelsolin or by release of the biologically active material beyond the stomach environment, such as in the intestine.

To ensure full gastric resistance a coating impermeable to at least pH 5.0 is essential.

Examples of the more common inert ingredients that are used as enteric coatings are cellulose acetate trimellitate (CAT), hydroxypropylmethylcellulose phthalate (HPMCP), HPMCP 50, HPMCP 55, polyvinyl acetate phthalate (PVAP), Eudragit L30D, Aquateric, cellulose acetate phthalate (CAP), Eudragit L, Eudragit S, and Shellac. These coatings may be used as mixed films.

A coating or mixture of coatings can also be used on tablets, which are not intended for protection against the stomach. This can include sugar coatings, or coatings which make the tablet easier to swallow. Capsules may consist of a hard shell (such as gelatin) for delivery of dry therapeutic i.e. powder; for liquid forms, a soft gelatin shell may be used. The shell material of cachets could be thick starch or other edible paper. For pills, lozenges, molded tablets or tablet triturates, moist massing techniques can be used.

The treatment or therapeutic agent can be included in the formulation as fine multi particulates in the form of granules or pellets of particle size about 1 mm. The formulation of the material for capsule administration could also be as a powder, lightly compressed plugs or even as tablets. The therapeutic could be prepared by compression.

Colorants and flavoring agents may all be included. For example, gelsolin may be formulated (such as by liposome or microsphere encapsulation) and then further contained within an edible product, such as a refrigerated beverage containing colorants and flavoring agents.

One may dilute or increase the volume of the therapeutic with an inert material. These diluents could include carbohydrates, especially mannitol, a lactose, anhydrous lactose, cellulose, sucrose, modified dextrans and starch. Certain inorganic salts may be also be used as fillers including calcium triphosphate, magnesium carbonate and sodium chloride. Some commercially available diluents are Fast-Flo, Emdex, STA-Rx 1500, Emcompress and Avicell.

Disintegrants may be included in the formulation of the therapeutic into a solid dosage form. Materials used as disintegrants include but are not limited to starch, including the commercial disintegrant based on starch, Explotab. Sodium starch glycolate, Amberlite, sodium carboxymethylcellulose, ultramylopectin, sodium alginate, gelatin, orange peel, acid carboxymethyl cellulose, natural sponge and bentonite may all be used. Another form of the disintegrants are the insoluble cationic exchange resins. Powdered gums may be used as disintegrants and as binders and these can include powdered gums such as agar, Karaya or tragacanth. Alginic acid and its sodium salt are also useful as disintegrants.

Binders may be used to hold the therapeutic agent together to form a hard tablet and include materials from natural products such as acacia, tragacanth, starch and gelatin. Others include methyl cellulose (MC), ethyl cellulose (EC) and carboxymethyl cellulose (CMC). Polyvinyl pyrrolidone (PVP) and hydroxypropylmethyl cellulose (HPMC) could both be used in alcoholic solutions to granulate the therapeutic.

An anti-frictional agent may be included in the formulation of the therapeutic to prevent sticking during the formulation process. Lubricants may be used as a layer between the therapeutic and the die wall, and these can include but are not limited to; stearic acid including its magnesium and calcium salts, polytetrafluoroethylene (PTFE), liquid paraffin, vegetable oils and waxes. Soluble lubricants may also be used such as sodium lauryl sulfate, magnesium lauryl sulfate, polyethylene glycol of various molecular weights, Carbowax 4000 and 6000.

Glidants that might improve the flow properties of the drug during formulation and to aid rearrangement during compression might be added. The glidants may include starch, talc, pyrogenic silica and hydrated silicoaluminate.

To aid dissolution of the therapeutic into the aqueous environment a surfactant might be added as a wetting agent. Surfactants may include anionic detergents such as sodium lauryl sulfate, dioctyl sodium sulfosuccinate and dioctyl sodium sulfonate. Cationic detergents might be used and could include benzalkonium chloride or benzethomium chloride. The list of potential non ionic detergents that could be included in the formulation as surfactants are lauromacrogol 400, polyoxyl 40 stearate, polyoxyethylene hydrogenated castor oil 10, 50 and 60, glycerol monostearate, polysorbate 40, 60, 65 and 80, sucrose fatty acid ester, methyl cellulose and carboxymethyl cellulose. These surfactants could be present in the formulation of gelsolin either alone or as a mixture in different ratios.

Pharmaceutical preparations which can be used orally include push fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.

Microspheres formulated for oral administration may also be used. Such

microspheres have been well defined in the art. All formulations for oral administration should be in dosages suitable for such administration.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by inhalation, the treatment or therapeutic agents for use according to the present disclosure may be conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g. gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

Also contemplated herein is pulmonary delivery of gelsolin. Gelsolin is delivered to the lungs of a mammal while inhaling and traverses across the lung epithelial lining to the blood stream. Other reports of inhaled molecules include Adjei et al., 1990, Pharmaceutical Research, 7:565 569; Adjei et al., 1990, International Journal of Pharmaceutics, 63: 135 144 (leuprolide acetate); Braquet et al., 1989, Journal of Cardiovascular Pharmacology, 13(suppl. 5): 143 146 (endothelin-1); Hubbard et al., 1989, Annals of Internal Medicine, Vol. Ill, pp. 206 212 (al antitrypsin); Smith et al., 1989, J. Clin. Invest. 84: 1145-1146 (a 1 -proteinase); Oswein et al., 1990, "Aerosolization of Proteins", Proceedings of Symposium on Respiratory Drug Delivery II, Keystone, Colorado, March, (recombinant human growth hormone); Debs et al., 1988, J. Immunol. 140:3482 3488 (interferon-γ and tumor necrosis factor alpha) and Platz et al., U.S. Patent No. 5,284,656 (granulocyte colony stimulating factor). A method and composition for pulmonary delivery of drugs for systemic effect is described in U.S. Patent No. 5,451,569, issued September 19, 1995 to Wong et al.

Contemplated for use in the practice of this disclosure are a wide range of mechanical devices designed for pulmonary delivery of therapeutic products, including but not limited to nebulizers, metered dose inhalers, and powder inhalers, all of which are familiar to those skilled in the art.

Some specific examples of commercially available devices suitable for the practice of this disclosure are the Ultravent nebulizer, manufactured by Mallinckrodt, Inc., St. Louis, Missouri; the Acorn II nebulizer, manufactured by Marquest Medical Products, Englewood, Colorado; the Ventolin metered dose inhaler, manufactured by Glaxo Inc., Research Triangle Park, North Carolina; and the Spinhaler powder inhaler, manufactured by Fisons Corp., Bedford, Massachusetts.

All such devices require the use of formulations suitable for the dispensing of gelsolin. Typically, each formulation is specific to the type of device employed and may involve the use of an appropriate propellant material, in addition to the usual diluents, adjuvants and/or carriers useful in therapy. Also, the use of liposomes, microcapsules or microspheres, inclusion complexes, or other types of carriers is contemplated. Chemically modified gelsolin may also be prepared in different formulations depending on the type of chemical modification or the type of device employed.

Formulations suitable for use with a nebulizer, either jet or ultrasonic, will typically comprise gelsolin dissolved in water at a concentration of about 0.1 to 25 mg of biologically active gelsolin per mL of solution. The formulation may also include a buffer and a simple sugar (e.g., for gelsolin stabilization and regulation of osmotic pressure). The nebulizer formulation may also contain a surfactant, to reduce or prevent surface induced aggregation of the gelsolin caused by atomization of the solution in forming the aerosol.

Formulations for use with a metered dose inhaler device will generally comprise a finely divided powder containing the gelsolin suspended in a propellant with the aid of a surfactant. The propellant may be any conventional material employed for this purpose, such as a chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol, and 1,1,1,2-tetrafluoroethane, or combinations thereof. Suitable surfactants include sorbitan trioleate and soya lecithin. Oleic acid may also be useful as a surfactant.

Formulations for dispensing from a powder inhaler device will comprise a finely divided dry powder containing gelsolin and may also include a bulking agent, such as lactose, sorbitol, sucrose, or mannitol in amounts which facilitate dispersal of the powder from the device, e.g., 50 to 90% by weight of the formulation. The gelsolin should most

advantageously be prepared in particulate form with an average particle size of less than 10 mm (or microns), most preferably 0.5 to 5 mm, for most effective delivery to the distal lung.

Nasal (or intranasal) delivery of a pharmaceutical composition of the present disclosure is also contemplated. Nasal delivery allows the passage of a pharmaceutical composition of the present disclosure to the blood stream directly after administering the therapeutic product to the nose, without the necessity for deposition of the product in the lung. Formulations for nasal delivery include those with dextran or cyclodextran.

For nasal administration, a useful device is a small, hard bottle to which a metered dose sprayer is attached. In one embodiment, the metered dose is delivered by drawing the pharmaceutical composition of the present disclosure solution into a chamber of defined volume, which chamber has an aperture dimensioned to aerosolize and aerosol formulation by forming a spray when a liquid in the chamber is compressed. The chamber is compressed to administer the pharmaceutical composition of the present disclosure. In a specific embodiment, the chamber is a piston arrangement. Such devices are commercially available.

Alternatively, a plastic squeeze bottle with an aperture or opening dimensioned to aerosolize an aerosol formulation by forming a spray when squeezed is used. The opening is usually found in the top of the bottle, and the top is generally tapered to partially fit in the nasal passages for efficient administration of the aerosol formulation. Preferably, the nasal inhaler will provide a metered amount of the aerosol formulation, for administration of a measured dose of the drug.

The treatment or therapeutic agents may also be formulated in rectal or vaginal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the treatment or therapeutic agents may also be formulated as a depot preparation. Such long acting formulations may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt. The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.

Suitable liquid or solid pharmaceutical preparation forms are, for example, aqueous or saline solutions for inhalation, microencapsulated, encochleated, coated onto microscopic gold particles, contained in liposomes, nebulized, aerosols, pellets for implantation into the skin, or dried onto a sharp object to be scratched into the skin. The pharmaceutical compositions also include granules, powders, tablets, coated tablets, (micro)capsules, suppositories, syrups, emulsions, suspensions, creams, drops or preparations with protracted release of active compounds, in whose preparation excipients and additives and/or auxiliaries such as disintegrants, binders, coating agents, swelling agents, lubricants, flavorings, sweeteners or solubilizers are customarily used as described above. The pharmaceutical compositions are suitable for use in a variety of drug delivery systems. For a brief review of methods for drug delivery, see Langer, Science 249: 1527-1533, 1990, which is incorporated herein by reference.

Gelsolin and optionally other therapeutics may be administered per se or in the form of a pharmaceutically acceptable salt.

The treatment or therapeutic agent(s), including specifically but not limited to gelsolin, may be provided in particles. Particles as used herein means nano or microparticles (or in some instances larger) which can consist in whole or in part of gelsolin or the other therapeutic agent(s) as described herein. The particles may contain the therapeutic agent(s) in a core surrounded by a coating, including, but not limited to, an enteric coating. The therapeutic agent(s) also may be dispersed throughout the particles. The therapeutic agent(s) also may be adsorbed into the particles. The particles may be of any order release kinetics, including zero order release, first order release, second order release, delayed release, sustained release, immediate release, and any combination thereof, etc. The particle may include, in addition to the therapeutic agent(s), any of those materials routinely used in the art of pharmacy and medicine, including, but not limited to, erodible, nonerodible,

biodegradable, or nonbiodegradable material or combinations thereof. The particles may be microcapsules which contain the gelsolin in a solution or in a semi-solid state. The particles may be of virtually any shape.

Both non-biodegradable and biodegradable polymeric materials can be used in the manufacture of particles for delivering the therapeutic agent(s). Such polymers may be natural or synthetic polymers. The polymer is selected based on the period of time over which release is desired. Bioadhesive polymers of particular interest include bioerodible hydrogels described by H.S. Sawhney, CP. Pathak and J.A. Hubell in Macromolecules, (1993) 26:581-587, the teachings of which are incorporated herein. These include polyhyaluronic acids, casein, gelatin, glutin, polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methyl methacrylates), poly(ethyl methacrylates), poly(butylmethacrylate), poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecyl methacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate), poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutyl acrylate), and poly(octadecyl acrylate).

The treatment or therapeutic agent(s) may be contained in controlled release systems. The term "controlled release" is intended to refer to any drug-containing formulation in which the manner and profile of drug release from the formulation are controlled. This refers to immediate as well as non-immediate release formulations, with non-immediate release formulations including but not limited to sustained release and delayed release formulations. The term "sustained release" (also referred to as "extended release") is used in its

conventional sense to refer to a drug formulation that provides for gradual release of a drug over an extended period of time, and that preferably, although not necessarily, results in substantially constant blood levels of a drug over an extended time period. The term "delayed release" is used in its conventional sense to refer to a drug formulation in which there is a time delay between administration of the formulation and the release of the drug therefrom. "Delayed release" may or may not involve gradual release of drug over an extended period of time, and thus may or may not be "sustained release."

Use of a long-term sustained release implant may be particularly suitable for treatment of chronic conditions. "Long-term" release, as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 7 days, and preferably 30-60 days. Long-term sustained release implants are well-known to those of ordinary skill in the art and include some of the release systems described above.

The disclosure also contemplates the use of kits. In some aspects of the disclosure, the kit can include a pharmaceutical preparation vial, a pharmaceutical preparation diluent vial, and gelsolin. The vial containing the diluent for the pharmaceutical preparation is optional. The diluent vial contains a diluent such as physiological saline for diluting what could be a concentrated solution or lyophilized powder of gelsolin. The instructions can include instructions for mixing a particular amount of the diluent with a particular amount of the concentrated pharmaceutical preparation, whereby a final formulation for injection or infusion is prepared. The instructions may include instructions for treating a subject with an effective amount of gelsolin. It also will be understood that the containers containing the preparations, whether the container is a bottle, a vial with a septum, an ampoule with a septum, an infusion bag, and the like, can contain indicia such as conventional markings which change color when the preparation has been autoclaved or otherwise sterilized.

The present disclosure is further illustrated by the following prophetic Examples, which in no way should be construed as further limiting. The entire contents of all of the references (including literature references, issued patents, published patent applications, and co pending patent applications) cited throughout this application are hereby expressly incorporated by reference.

Examples

Example 1

pGSN levels is determined in subjects with SCD at steady state and correlated with hemolysis parameters that are consistently abnormal in SCD: anemia, high reticulocyte count, and elevated serum bilirubin and LDH concentrations. We predict that lower pGSN levels will correlate with worse anemia and higher amounts of the other measurements.

Example 2

pGSN is administered to Berkeley sickle cell mice that produce red cells containing human sickle cell hemoglobin and have brisk hemolysis (Manci et al, Pathology of Berkeley sickle cell mice: similarity to human sickle cell disease. Blood 107: 1651-1658, 2007). We predict that this treatment will alter the hemolysis parameters summarized above in a manner consistent with decreased hemolysis.

Example 3

Summary:

Plasma gelsolin levels (pGSN) are believed to provide therapeutic and diagnostic value in sickle cell disease (SCD). pGSN levels were measured in plasma of sickle cell patients using sandwich ELISA methodology. Plasma samples were obtained from twelve individuals greater than four years of age with a known diagnosis of sickle cell disease (all genotypes) who are at baseline (in steady state). Plasma gelsolin samples in these patients varied widely and on average were well below those found in normal healthy individuals. In two patients whose clinical profile was examined, plasma gelsolin levels correlated with severity of disease.

Materials and Methods:

Patient samples

The patients with sickle cell disease undergo frequent, routine blood testing. This study was completed in conjunction with patients' routine blood draws.

Inclusion criterion: Individuals greater than four years of age with a known diagnosis of sickle cell disease (all genotypes) who are at baseline (in steady state).

Exclusion criteria:

-People who are not undergoing routine blood draw or whose parents refuse their

participation.

-Any patient who has received a blood transfusion within 30 days.

-Any patient who does not wish to participate.

-For parents, patients, and individuals who cannot read, consent is obtained via verbal description by the research assistant and verified by the study principal investigator (PI).

Procedure:

Two mis of blood anticoagulated with EDTA were drawn in conjunction with routine blood draws in patients with SCD for baseline assessment of plasma Gelsolin (pGSN). These levels were drawn at steady-state (i.e. when the patient is feeling well).

All samples were labeled only with the participant study ID number and were collected from the clinic by the PI, research assistant, or nurse coordinator.

The samples were centrifuged, and the supernatant fluids removed and quick frozen. The frozen EDTA plasmas were stored at -70°C and shipped in batches to BioAegis Therapeutics laboratory in New Jersey.

Normal Healthy Individual Samples

Plasma from eight normal healthy individuals was obtained from Innovative Research, Novi, ML

pGSN was assayed by sandwich ELISA. Blinded patient samples were compared to the standard curve using known concentrations of pure recombinant human plasma gelsolin.

Results: ELISA values of plasma levels of pGSN were evaluated based on a standard curve generated with known amounts of recombinant human plasma gelsolin (rhu-pGSN) (Figure 1).

Samples from patients were evaluated and data is shown in Table 1 below. Sickle cell patients' plasma levels of pGSN were found to be from 50% to 107% compared to a group of normal healthy individuals. Overall, the average plasma gelsolin level was 75% of the level of the normal cohort. Plasma gelsolin levels in these patients vary as compared to normal healthy individuals. The two patients with the highest (POC127) and lowest (POCl 18) values were unblinded by the PI to assess their clinical profile.

Patient POC 127 who was found to have normal levels of plasma gelsolin is a stable patient, effectively controlled on currently available treatment for sickle cell disease. Patient POCl 18 who was found to have the lowest level of plasma gelsolin in the patient cohort and compared to normal controls is a poorly controlled patient subject to frequent exacerbations of the disease process. of Normal Average of

Sample Average Std Dev :CV Normal Sample three runs: Std Dev CV

POC10_{4 4}0.798 2.258! 6%! 71%

POC109 33.82₄ 3.753: 11%! 59% LS03 62.705 5.7413 9%

POC110 55.799 3.160: 6%: 98% LS04 60.427 6.463085 11%

POC111 ₄9.762 4.829: 10%: 87% LS06 53.073 4.78293 9%

POC112 ₄3.073 2.664! 6%! 75% LS09 58.782 4.748585 8%

POC113 37.79₄ 1.623! 4%! 66% LS10 48.951 4.207575 9%

POC116 34171 1.392! 4%! 60% LS32 57.959 7.255486 13%

POC117 35.320 2.568! 7%: 62% LS37 57.646 10.63148 18%

POC118 28.556 0.730! 3%! 50% LS42 57.990 8.464948 15%

POC121 ₄6.551 0.109! 0%! 81%

POC126 55.₄2₄ 16.984! 31%: 97% Average: 57.192

POC127 61.361 5.688! 9%: 107%

Average: 43.536

Table 1. Results of determination of plasma levels of pGSN in sickle cell patients (POC) as compared to normal controls (LS).

Equivalents

The foregoing written specification is considered to be sufficient to enable one ordinarily skilled in the art to practice the disclosure. The present disclosure is not to be limited in scope by examples provided, since the examples are intended as mere illustrations of one or more aspects of the disclosure. Other functionally equivalent embodiments are considered within the scope of the disclosure. Various modifications of the disclosure in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description. Each of the limitations of the disclosure can encompass various embodiments of the disclosure. It is, therefore, anticipated that each of the limitations of the disclosure involving any one element or combinations of elements can be included in each aspect of the disclosure. This disclosure is not limited in its application to the details of construction and the arrangement of components set forth or illustrated in the drawings. The disclosure is capable of other embodiments and of being practiced or of being carried out in various ways.

Also, the phraseology and terminology used herein is for the purpose of description and should not be regarded as limiting. The use of "including," "comprising," or "having," "containing", "involving", and variations thereof herein, is meant to encompass the items listed thereafter and equivalents thereof as well as additional items.

All references, patents and patent applications that are recited in this application are incorporated by reference herein in their entirety.

We claim:

Claims

Claims

1. A method of treating a subject having or at risk of developing a Sickle Cell Disease (SCD) complication, the method comprising:

administering to a subject in need of such a treatment an effective amount of gelsolin to treat the subject.

2. A method of treating a subject having or at risk of developing a Sickle Cell Disease (SCD) complication to reduce the risk of the SCD complication, the method comprising: administering to a subject in need of such a treatment an effective amount of gelsolin to raise the level of gelsolin in the subject above a reference level.

3. The method of claim 1 or claim 2, wherein the subject is otherwise free of indications calling for treatment with gelsolin.

4. The method of any one of claims 1-3, wherein the gelsolin is plasma gelsolin (pGSN) or cytoplasmic gelsolin (cGSN).

5. The method of any one of claims 1-4, wherein the gelsolin is administered orally, sublingually, buccally, intranasally, intravenously, intramuscularly, intrathecally, intraperitoneally, subcutaneously, or by inhalation.

6. The method of any one of claims 1-5, wherein the gelsolin is administered prophylactically.

7. The method of any one of claims 1-6, further comprising administering a second agent for treating a SCD complication.

8. The method of claim 7, wherein the second agent is hydroxyurea, folic acid, an iron chelator, heparin, pentosan polysulfate, diphenhydramine, nitric oxide, L-arginine, eptifibatide, prasugrel, zileuton (Zyflo CR), a statin, a corticosteroid, a glucocorticoid, regadenoson (Lexiscan™), 5-hydroxymethyl-2-furfural (Aes-103), a selectin antagonist, a selectin antibody, humanized antibody SelGl, Rivipansel, an integrin antagonist, an integrin antibody, an analgesic, an anti-inflammatory agent, oxygen inhalation, intravenous hydration, or a blood transfusion.

9. The method of any one of claims 1-8, further comprising having the subject undergo an exchange transfusion.

10. A method of treating a subject at an increased risk of developing a Sickle Cell Disease (SCD) complication to reduce the risk of the SCD complication, the method comprising: selecting a subject on the basis that the subject is known to have a level of gelsolin below a reference level and

administering to the subject gelsolin and/or a second agent in an effective amount to reduce the subject's risk of developing a SCD complication.

11. A method of treating a subject at an increased risk of developing a Sickle Cell Disease (SCD) complication to reduce the risk of a SCD complication, the method comprising:

selecting the subject on the basis that the subject is known to have a level of gelsolin below a reference level and

administering to the subject gelsolin and/or a second agent in an effective amount to raise the level of gelsolin in the subject above a reference level.

12. The method of claim 10 or claim 11, wherein the subject is otherwise free of indications calling for treatment with gelsolin.

13. The method of any one of claims 10-12, wherein the subject is apparently healthy.

14. The method of any one of claims 10-13, wherein the gelsolin is plasma gelsolin (pGSN) or cytoplasmic gelsolin (cGSN).

15. The method of any one of claims 10-12, further comprising administering a second agent for treating a SCD complication.

16. The method of claim 15, wherein the second agent is hydroxyurea, folic acid, an iron chelator, heparin, pentosan polysulfate, diphenhydramine, nitric oxide, L-arginine, eptifibatide, prasugrel, zileuton (Zyflo CR), a statin, a corticosteroid, a glucocorticoid, regadenoson (Lexiscan™), 5-hydroxymethyl-2-furfural (Aes-103), a selectin antagonist, a selectin antibody, humanized antibody SelGl, Rivipansel, an integrin antagonist, an integrin antibody, an analgesic, an anti-inflammatory agent, oxygen inhalation, intravenous hydration, or a blood transfusion.

17. Gelsolin for use in a method for treating a subject having or at risk of developing a SCD complication.

18. The gelsolin of claim 17, wherein the subject is otherwise free of indications calling for treatment with gelsolin.

19. The gelsolin of claim 17 or 18, wherein the gelsolin is plasma gelsolin (pGSN) or cytoplasmic gelsolin (cGSN).

20. The gelsolin of any one of claims 17-19, wherein the gelsolin is for administration orally, sublingually, buccally, intranasally, intravenously, intramuscularly, intrathecally, intraperitoneally, subcutaneously, or by inhalation.

21. The gelsolin of any one of claims 17-20 for use with a second agent wherein the second agent is hydroxyurea, folic acid, an iron chelator, heparin, pentosan polysulfate, diphenhydramine, nitric oxide, L-arginine, eptifibatide, prasugrel, zileuton (Zyflo CR), a statin, a corticosteroid, a glucocorticoid, regadenoson (Lexiscan™), 5-hydroxymethyl-2- furfural (Aes-103), a selectin antagonist, a selectin antibody, humanized antibody SelGl, Rivipansel, an integrin antagonist, an integrin antibody, an analgesic, an anti-inflammatory agent, oxygen inhalation, intravenous hydration, or a blood transfusion.

22. Gelsolin for use in a method for treating a subject having or at risk of developing a SCD complication wherein the subject is selected on the basis that the subject is known to have a level of gelsolin below a reference level.

23. The gelsolin of claim 22, wherein the subject is otherwise free of indications calling for treatment with gelsolin.

24. The gelsolin of claim 22 or 23, wherein the gelsolin is plasma gelsolin (pGSN) or cytoplasmic gelsolin (cGSN).

25. The gelsolin of any one of claims 22-24,wherein the gelsolin is for administration orally, sublingually, buccally, intranasally, intravenously, intramuscularly, intrathecally, intraperitoneally, subcutaneously, or by inhalation.

26. The gelsolin of any one of claims 22-25 for use with a second agent wherein the second agent is hydroxyurea, folic acid, an iron chelator, heparin, pentosan polysulfate, diphenhydramine, nitric oxide, L-arginine, eptifibatide, prasugrel, zileuton (Zyflo CR), a statin, a corticosteroid, a glucocorticoid, regadenoson (Lexiscan™), 5-hydroxymethyl-2- furfural (Aes-103), a selectin antagonist, a selectin antibody, humanized antibody SelGl, Rivipansel, an integrin antagonist, an integrin antibody, an analgesic, an anti-inflammatory agent, oxygen inhalation, intravenous hydration, or a blood transfusion.

27. The use of gelsolin in the manufacture of a medicament for treating a subject having or at risk of developing a SCD complication.

28. The use of claim 27, wherein the subject is otherwise free of indications calling for treatment with gelsolin.

29. The use of claim 27 or 28, wherein the gelsolin is plasma gelsolin (pGSN) or cytoplasmic gelsolin (cGSN).

30. The use of any one of claims 27-29, wherein the gelsolin is for administration orally, sublingually, buccally, intranasally, intravenously, intramuscularly, intrathecally,

intraperitoneally, subcutaneously, or by inhalation.

31. The use of any one of claims 27-30 with a second agent wherein the second agent is hydroxyurea, folic acid, an iron chelator, heparin, pentosan polysulfate, diphenhydramine, nitric oxide, L-arginine, eptifibatide, prasugrel, zileuton (Zyflo CR), a statin, a corticosteroid, a glucocorticoid, regadenoson (Lexiscan™), 5-hydroxymethyl-2-furfural (Aes-103), a selectin antagonist, a selectin antibody, humanized antibody SelGl, Rivipansel, an integrin antagonist, an integrin antibody, an analgesic, or an anti-inflammatory agent.

32. The use of gelsolin in the manufacture of a medicament for treating a subject having or at risk of developing a SCD complication wherein the subject is selected on the basis that the subject is known to have a level of gelsolin below a reference level.

33. The use of claim 32, wherein the subject is otherwise free of indications calling for treatment with gelsolin.

34. The use of claim 32 or 33, wherein the gelsolin is plasma gelsolin (pGSN) or cytoplasmic gelsolin (cGSN).

35. The use of any one of claims 32-34,wherein the gelsolin is for administration orally, sublingually, buccally, intranasally, intravenously, intramuscularly, intrathecally,

intraperitoneally, subcutaneously, or by inhalation.

36. The use of any one of claims 32-35 with a second agent wherein the second agent is hydroxyurea, folic acid, an iron chelator, heparin, pentosan polysulfate, diphenhydramine, nitric oxide, L-arginine, eptifibatide, prasugrel, zileuton (Zyflo CR), a statin, a corticosteroid, a glucocorticoid, regadenoson (Lexiscan™), 5-hydroxymethyl-2-furfural (Aes-103), a selectin antagonist, a selectin antibody, humanized antibody SelGl, Rivipansel, an integrin antagonist, an integrin antibody, an analgesic, or an anti-inflammatory agent.

37. A method of characterizing a subject's risk of developing a Sickle Cell Disease (SCD) complication, the method comprising:

comparing a level of gelsolin in a sample from the subject to a reference level, and characterizing the subject's risk of developing a SCD complication based upon the level of gelsolin in comparison to the reference level, wherein a level of gelsolin below the reference level is indicative that the subject is at an elevated risk of developing a SCD complication, and/or wherein a level of gelsolin at or above the reference level is indicative that the subject is not at an elevated risk of developing a SCD complication.

38. The method of claim 37, further comprising determining the level of gelsolin in the sample.

39. The method of claim 37 or claim 38, further comprising providing a report indicating that the subject is at an elevated risk of developing a SCD complication when the level of gelsolin is below the reference level.

40. The method of any one of claims 37-39, further comprising providing a report indicating that the subject is not at an elevated risk of developing a SCD complication when the level of gelsolin is at or above the reference level.

41. The method of any one of claims 37-40, wherein the gelsolin is plasma gelsolin (pGSN) or cytoplasmic gelsolin (cGSN).

42. The method of any one of claims 37-41, wherein the level of gelsolin is in a body fluid of the subject.

43. The method of claim 42, wherein the body fluid is blood, plasma, serum, urine, or saliva.

44. The method of any one of claims 37-43, wherein the reference level is about 250 mg/L of plasma.

45. The method of any one of claims 37-44, wherein the subject is an apparently healthy subject.

46. The method of any one of claims 37-45, further comprising performing one or more tests to evaluate a SCD complication.

47. The method of claim 46, wherein the test is a blood reticulocyte count, blood smear inspection, hemoglobin level, hemoglobin electrophoresis, hemoglobin S, or sickle cell index.

48. A method of evaluating the efficacy of a treatment of a Sickle Cell Disease (SCD) complication in a subject, the method comprising:

(i) determining a level of gelsolin in a sample from the subject undergoing treatment with an agent to treat or reduce the risk of a SCD complication, (ii) comparing the level of gelsolin obtained in (i) to a reference level corresponding to a level of gelsolin in an apparently healthy control population, and

(iii) determining whether the level of gelsolin in (i) is at or above the reference level, said determination being indicative that the treatment is efficacious.

49. The method of claim 48, wherein step (i) and step (ii) and /or step (iii) are repeated so as to monitor the level(s) of gelsolin over time.

50. The method of claim 48 or claim 49, wherein the reference level is about 250 mg/L of plasma.

51. The method of any of claims 48-50, wherein the treatment is gelsolin, hydroxyurea, folic acid, an iron chelator, heparin, pentosan polysulfate, diphenhydramine, nitric oxide, L- arginine, eptifibatide, prasugrel, zileuton (Zyflo CR), a statin, a corticosteroid, a

glucocorticoid, regadenoson (Lexiscan™), 5-hydroxymethyl-2-furfural (Aes-103), a selectin antagonist, a selectin antibody, humanized antibody SelGl, Rivipansel, an integrin antagonist, an integrin antibody, an analgesic, an anti-inflammatory agent, oxygen inhalation, intravenous hydration, or a blood transfusion.

52. A method of deciding on the course of a treatment in a subject, the method

comprising:

(i) determining a level of gelsolin in a sample from the subject undergoing a treatment of a Sickle Cell Disease (SCD) complication,

(ii) comparing the level of gelsolin obtained in (i) to a reference level corresponding to a level of gelsolin corresponding to a level of gelsolin in an apparently healthy control population,

(iii) determining whether the level of gelsolin obtained in (i) is at or below the reference level, and

(iv) deciding on the course of the treatment based on such determination.

53. The method of claim 52, wherein step (i) and step (ii) and /or step (iii) are repeated so as to monitor the level(s) of gelsolin over time.

54. The method of claim 52 or claim 53, wherein the reference level is about 250 mg/L of plasma or lower.