WO2012109732A1 - Compositions comprising self-assembling peptide and amino acid vehicles and active agents pp1 or pp2 and uses thereof - Google Patents

Abstract

The present disclosure relates to 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo[3,4-d]-pyrimidine ("PP1") or (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine ("PP2") drug vehicles comprising a self-assembling peptide in combination with a single amino acid, pharmaceutical compositions comprising PP1 or PP2 and the vehicle and methods and uses thereof.

Description

COMPOSITIONS COMPRISING SELF-ASSEMBLING PEPTIDE AND AMINO ACID VEHICLES AND ACTIVE AGENTS PP1 OR PP2 AND USES THEREOF

RELATED APPLICATIONS

[0001] This application claims the benefit of priority of copending U.S. provisional application No. 61/443,833 filed February 17, 2011 , the contents of which are incorporated herein by reference in their entirety.

FIELD

[0002] The present disclosure relates to compositions and drug delivery vehicles comprising self-assembling peptides in combination with single amino acids and methods and uses thereof in delivery of a chemical inhibitor of the Src tyrosine kinase family.

BACKGROUND

[0003] Advances in nanobiomaterials have tremendous impacts on contemporary medicine, especially in drug delivery and tissue repair. These applications rely on the development of (nano)materials with good biocompatibility, bio-safety and bio-activity. Among many new nanobiomaterials, a special class of self-assembling peptides has been extensively explored for tissue engineering (Zhang, 2003). These peptides have unique repetitive amino acid sequences that allow them to self- assemble into stable nanostructures. More importantly, they have good biocompatibility and are biodegradable; no immune responses were observed when introduced into animals (Zhang et al., 1993; Zhang et al., 1995). These properties make them ideal biomaterials for uses in drug delivery and regenerative medicine. Recently, these peptides have been used as nano- carriers for the delivery of hydrophobic molecules and siRNA (Fung et al., 2008; Fung et al., 2009; Law et al., 2008).

[0004] The self-assembling peptides have long been applied as templates for tissue regeneration in vitro and in vivo (Davis et al., 2005; Ellis- Behnke et al., 2006; Holmes et al., 2000; Zhang et al., 1995). These peptides are highly compatible with mammalian cells and tissues, and display minimum immune responses in vivo; hence, they hold great promise as new clinically applicable biomaterials.

[0005] Amino acids are the fundamental molecules in all life forms. They serve as nutrients and building units to construct functional biomolecules. These naturally existing molecules are also widely used in many biotechnology and pharmaceutical applications. They are primarily employed as solvent additives for protein purification and excipients for protein formulation (Arakawa et al., 2007). Amino acids are currently used in clinical infusion for many clinical applications, such as nutrient supplies and treatments for some liver diseases. They have been employed in protein purification or as excipients for protein formulation to increase the biocompatibility, osmotic pressure and protein stability (Arakawa et al., 2007; Chen et al., 2003).

[0006] Acute lung injury (ALI) often results from uncontrolled, excessive inflammatory responses. Although many efforts have been put in clinical trials to treat such a problem, the outcome is still unsatisfactory (the mortality has not decreased substantially since 1994 (Phua et al., 2009)). Recently, Src tyrosine kinase signaling pathways have been found to play critical roles in acute inflammatory responses (Okutani et al., 2006). They are involved in recruitment and activation of various immune cells, and in regulation of vascular permeability and tissue inflammation. Thus, inhibition of Src signaling has been explored as a new strategy to treat acute inflammatory responses related diseases.

[0007] For such a purpose, one class of chemical inhibitors (PP1/PP2) has been discovered to selectively inhibit Src tyrosine kinase family (Hanke et al., 1996). Later, several reports have shown that these Src inhibitors are capable of reducing acute inflammatory responses in many disease animal models, including acute lung injury, brain injury, stroke and myocardial infarction (Okutani et al., 2006; Khadaroo et al., 2004; Severgnini et al., 2005; Lee et al., 2007). The present inventors have recently also demonstrated that PP2 can protect lungs from ischemia-reperfusion induced acute injury in a rat model (Oyaizu et al., 2012).

SUMMARY

[0008] The present inventors have developed a nanoscale formulation using the combination of self-assembling peptides with amino acids to deliver a Src PTK inhibitor: 4-amino-5-(4-methylphenyl)-7-(f-butyl)pyrazolo[3,4-d]- pyrimidine ("PP1") or (4-amino-5-(4-chlorophenyl)-7-(f-butyl)pyrazolo[3,4- djpyrimidine ("PP2"). As an example, PP2 was formulated using self- assembling peptides in combination with amino acids to improve the formulation biocompatibility and safety for potential clinical uses. The nanoformulations were compared with those containing large amounts of the organic solvent dimethyl sulfoxide (DMSO) regarding their hemolytic activity and acute in vivo toxicity. The objective was to reduce the use of DMSO by introducing non-toxic, clinically applicable materials while maintaining the drug solubility and its therapeutic efficacy. A lipopolysaccharide (LPS)-induced ALI mouse model was then established to evaluate the anti-inflammatory effect of the PP2 nanoformulations in vivo.

[0009] The nanoscale formulation strategy developed using the combination of the self-assembling peptide EAK16-II with single amino acids improves the biocompatibility of the PP2 formulation for clinical uses. The developed PP2 nanoformulations are non-hemolytic, non-toxic (acute phase), active in Src inhibition on cultured cancer cells, and effective in anti- inflammation on LPS-induced acute lung injury mouse model.

[0010] Accordingly, the present disclosure provides a vehicle for delivering 4-amino-5-(4-methylphenyl)-7-(i-butyl)pyrazolo[3,4-o -pyrimidine ("ΡΡΓ) or (4-amino-5-(4-chlorophenyl)-7-(i-butyl)pyrazolo[3,4-c/]pyrimidine ("PP2") comprising a self-assembling peptide consisting of lysine, glutamate and alanine amino acid residues in combination with a single amino acid.

[0011] The single amino acid can be any amino acid. In one embodiment, the amino acid is leucine, phenylalanine, tryptophan, or methionine. In another embodiment, the amino acid is alanine, isoleucine or asparagine. In yet another embodiment, the amino acid is glutamic acid or aspartic acid.

[0012] In one embodiment, the self-assembling peptide comprises 12- 30 amino acid residues. In one embodiment, the self-assembling peptide consisting of lysine, glutamate and alanine or EAK is EAK16-I, EAK16-II or EAK16-IV.

[0013] The vehicle further comprises less than 20% co-solvent, less than 15%, less than 10%, less than 5% or less than 1% co-solvent. In an embodiment, the vehicle comprises 0.5 to 10% co-solvent, 0.5 to 5% co- solvent or 0.5 to 1% co-solvent. In one embodiment, the co-solvent is DMSO or ethanol.

[0014] Further provided herein is a pharmaceutical composition comprising 4-amino-5-(4-methylphenyl)-7-(/-butyl)pyrazolo[3,4-c]-pyrimidine ("PP1") or (4-amino-5-(4-chlorophenyl)-7-(/-butyl)pyrazolo[3,4-c]pyrimidine ("PP2") combined with the vehicle disclosed herein.

[0015] Also provided herein is a method of delivering 4-amino-5-(4- methylphenyl)-7-(i-butyl)pyrazolo[3,4-c ]-pyrimidine ("PP1") or (4-amino-5-(4- chlorophenyl)-7-(i-butyl)pyrazolo[3,4- /]pyrimidine ("PP2") to a cell or animal in need thereof comprising administering the composition disclosed herein to the cell or animal. In another embodiment, the present disclosure provides use of the composition disclosed herein for delivery of PP1 or PP2 to a cell or animal in need thereof. Also provided herein is use of the composition disclosed herein in the manufacture of a medicament for delivery of PP1 or PP2 to a cell or animal in need thereof. Further provided is a composition disclosed herein for use in delivering PP1 or PP2 to a cell or animal in need thereof. In one embodiment, the method is for treating cancer or an acute inflammatory disease or acute inflammatory condition. [0016] The composition can be administered by any suitable method, including by inhalation, intratracheal administration, or intravenous administration.

[0017] Also provided herein is a method of treating cancer comprising administering a pharmaceutical composition comprising 4-amino-5-(4- methylphenyl)-7-(i-butyl)pyrazolo[3,4- /]-pyrimidine ("PP1") or (4-amino-5-(4- chlorophenyl)-7-(f-butyl)pyrazolo[3,4-alpyrimidine ("PP2") in combination with the vehicle disclosed herein to a subject in need thereof. In another embodiment, the present disclosure provides a use of a pharmaceutical composition comprising PP1 or PP2 in combination with the vehicle disclosed herein for treating cancer. Also provided herein is a use of PP1 or PP2 in combination with the vehicle disclosed herein in the preparation of a medicament for treating cancer. Further provided herein is a pharmaceutical composition comprising PP1 or PP2 in combination with the vehicle disclosed herein for use in treating cancer.

[0018] In yet another embodiment, the present disclosure provides a method of treating acute inflammatory disease or acute inflammatory condition comprising administering a pharmaceutical composition comprising 4-amino-5-(4-methylphenyl)-7-(i-butyl)pyrazolo[3,4-c]-pyrimidine ("PP1") or (4-amino-5-(4-chlorophenyl)-7-(f-butyl)pyrazolo[3,4-d]pyrimidine ("PP2") in combination with the vehicle disclosed herein to a subject in need thereof. In another embodiment, the present disclosure provides a use of a pharmaceutical composition comprising PP1 or PP2 in combination with the vehicle disclosed herein for treating acute inflammatory disease or acute inflammatory condition. Also provided herein is a use of PP1 or PP2 in combination with the vehicle disclosed herein in the preparation of a medicament for treating acute inflammatory disease or acute inflammatory condition. Further provided herein is a pharmaceutical composition comprising PP1 or PP2 in combination with the vehicle disclosed herein for use in treating acute inflammatory disease or acute inflammatory condition. In one embodiment, the acute inflammatory disease or acute inflammatory condition is acute lung injury, brain injury, stroke, myocardial infarction or ischemia-reperfusion induced acute injury.

[0019] In one embodiment, the compositions, methods and uses disclosed herein comprise PP2. In another embodiment, the compositions, methods and uses disclosed herein comprise PP1.

[0020] Further provided herein is a method of preparing a composition of 4-amino-5-(4-methylphenyl)-7-(i-butyl)pyrazolo[3,4-oi]-pyrimidine ("PP1") or (4-amino-5-(4-chlorophenyl)-7-(f-butyl)pyrazolo[3,4-c ]pyrimidine ("PP2") with reduced solvent concentration comprising combining PP1 or PP2 with the vehicle disclosed herein. In one embodiment, the reduced solvent is DMSO or ethanol.

[0021] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples while indicating preferred embodiments of the disclosure are given by way of illustration only, since various changes and modifications within the spirit and scope of the disclosure will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022] The disclosure will now be described in relation to the drawings in which:

[0023] Figure 1 shows PP2 formulations in DMSO/saline for intravenous injection and their toxicity. (A) A minimum of 20% DMSO is required to dissolve 0.05 mg/ml PP2 in saline. (B) Injection of large amount of DMSO in tail vein causes hemolysis.

[0024] Figure 2 shows the molecular structures of the formulation molecules and the formulation strategy.

[0025] Figure 3 shows solubilization of 0.05 mg/ml PP2 with different formulations: (A) 0.01 mg/ml EAK16-II with DMSO (1 -20%); (B) four different amino acids with 1 % DMSO; (C) the combinations of 0.1 mg/ml EAK16-II with single amino acids at 1% DMSO; the amino acid concentrations were indicated in the parentheses (mg/ml).

[0026] Figure 4 shows ex vivo hemolytic activity studies of PP2 nanoformulations. (A) Direct observation of hemolysis and red blood cell (RBC) aggregation; 1-5 represents serial sample dilutions (low to high formula concentration); + pure water; - saline. (B) RBC aggregations observed by optical imaging.

[0027] Figure 5 shows inhibition of Src activity by PP2 nanoformulations on cultured cells A549 (A) and v-Src transformed NIH 3T3 (B-D). (A and B) Immunoblotting shows inhibition of total tyrosine phosphorylation by the PP2 nanoformulation; (C) immunoblotting of STAT3 phosphorylation, a down-stream signal of Src kinase; (D) change in cell morphology after 24 h PP2 treatment on Src inhibition. PP2 final concentration in the medium: 10 μΜ.

[0028] Figure 6 shows evaluation of in vivo safety of the nanoformulation on the LPS-induced lung injury model. (A) The model scheme; (B) the 24 h survival curve of the PP2-nanoformulation in comparison with the control (PP2-20%DMSO); (C) the histology of the lung tissues from the mice suffered immediate death (in 5 min) upon the PP2- 20%DMSO treatment via intratracheal instillation; images were taken at 400 x magnification.

[0029] Figure 7 shows evaluation of the anti-inflammatory effect of the nanoformulations on the LPS-induced lung injury mouse model. All readouts were from the BALF. (A) Total cell counts; (B) the ratio of the neutrophils (NP) to macrophages (AM); (C) total protein concentration; (D) pro-inflammatory cytokine TNF- level; (E) regulatory cytokine IL-10 level. * p < 0.05; ** p <0.01. Error bar represents SE.

DETAILED DESCRIPTION [0030] Many newly discovered therapeutic agents require a delivery platform in order to translate them into clinical applications. As an example, a nanoscale formulation strategy was developed for the Src tyrosine kinase inhibitor PP2. The formulation utilized the combination of the self-assembling peptides (EAK16-II) and amino acids to minimize the use of the toxic organic solvent DMSO; hence, the biocompatibility of the PP2 nanoformulations was significantly improved. They were found to be non-hemolytic and safe for intravenous and intratracheal administration; the formulations did not alter PP2 activity in Src inhibition on cultured cells. The PP2 nanoformulation was further evaluated on a lipopolysaccharide (LPS)-induced acute lung injury mouse model. Results revealed that the pretreatment of PP2 nanoformulation could decrease the inflammatory cell infiltration and the pro-inflammatory cytokine TNF-a production in the bronchoalveolar lavage fluid after LPS stimulation.

[0031] Accordingly, the present disclosure provides a vehicle for delivering 4-amino-5-(4-methylphenyl)-7-(f-butyl)pyrazolo[3,4-d]-pyrimidine ("PP1") or (4-amino-5-(4-chlorophenyl)-7-(/-butyl)pyrazolo[3,4-c]pyrimidine ("PP2") comprising a self-assembling peptide consisting of lysine, glutamate and alanine amino acid residues in combination with a single amino acid.

[0032] As used herein, the term "amino acid" refers to a molecule consisting of a carbon atom to which are attached an amino group, a carboxylic acid group, and a side chain which varies among the different amino acids. For example, the term "amino acid" includes, but is not limited to, the 20 basic building blocks of a protein: Arginine, Histidine, Lysine, Glycine, Alanine, Valine, Leucine, Isoleucine, Serine, Threonine, Asparagine, Glutamine, Cysteine, Proline, Methionine, Phenylalanine, Tryptophan, Tyrosine, Aspartic acid and Glutamic acid. Additionally, as used herein, "amino acid" also includes derivatives of amino acids such as esters, and amides, and salts, as well as other derivatives and non-standard amino acids. [0033] The single amino acid disclosed herein can be any amino acid that provides solubility of the formulation at the desired concentration. In one embodiment, the amino acid is leucine, phenylalanine, tryptophan, or methionine. In another embodiment, the amino acid is alanine, isoleucine or asparagine. In yet another embodiment, the amino acid is glutamic acid or aspartic acid.

[0034] The term "self-assembling peptide" as used herein refers to peptides, optionally 12-30 amino acids in length, which are able to assemble into stable membranes and filaments. Such self-assembling peptides include ionic complementary peptides and surfactant-like peptides. Self-assembling peptides permit the hydrophobic compound, PP1 or PP2, to be more or less surrounded by an amphiphilic structure, thereby becoming, as a unit, more water soluble.

[0035] Accordingly, in one embodiment, the self-assembling peptide comprises 12-30 amino acid residues, optionally 14-20 amino acids. In a particular embodiment, the self-assembling peptide is 16 amino acids.

[0036] In one embodiment, the self-assembling peptide consisting of lysine, glutamate and alanine or EAK is EAK16-I (AEAKAEAKAEAKAEAK (SEQ ID NO:1 )), EAK16-II (AEAEAKAKAEAEAKAK (SEQ ID NO:2)) or EAK16-IV (AEAEAEAEAKAKAKAK (SEQ ID NO:3)).

[0037] The peptides disclosed herein may be prepared using recombinant DNA methods. These peptides may be purified and/or isolated to various degrees using techniques known in the art. Accordingly, nucleic acid molecules having a sequence which encodes a self-assembling peptide may be incorporated according to procedures known in the art into an appropriate expression vector which ensures good expression of the peptide. Possible expression vectors include but are not limited to cosmids, plasmids, or modified viruses (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), so long as the vector is compatible with the host cell used. The expression "vectors suitable for transformation of a host cell", means that the expression vectors contain a nucleic acid molecule and regulatory sequences, selected on the basis of the host cells to be used for expression, which are operatively linked to the nucleic acid molecule. "Operatively linked" is intended to mean that the nucleic acid is linked to regulatory sequences in a manner which allows expression of the nucleic acid.

[0038] Suitable host cells include a wide variety of prokaryotic and eukaryotic host cells. For example, the proteins of the disclosure may be expressed in bacterial cells such as E. coli, insect cells (using baculovirus), yeast cells or mammalian cells, COS1 cells. Other suitable host cells can be found in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1991 ).

[0039] The peptides can be isolated from a host cell expressing the peptide and purified according to standard procedures of the art, including ammonium sulfate precipitation, column chromatography (e.g. ion exchange, gel filtration, affinity chromatography, etc.), electrophoresis, and ultimately, crystallization [see generally, "Enzyme Purification and Related Techniques", Methods in Enzymology, 22, 233-577 (1971 )].

[0040] Alternatively, the peptide can be prepared by chemical synthesis using techniques well known in the chemistry of proteins such as solid phase synthesis (Merrifield, 1963) or synthesis in homogeneous solution (Houbenwycl, 1987).

[0041] Typically, hydrophobic compositions need to be formulated with solvents for solubility of the compound. Typically the majority of solvent is water. The presence of co-solvents limits the bioavailability of such compounds and causes toxicity. The present disclosure provides for compositions with reduced need for co-solvents resulting in a reduced toxicity. Accordingly, in an embodiment, the vehicle further comprises less than 20% co-solvent, less than 15%, less than 10%, less than 5% or less than 1 % co- solvent. In an embodiment, the vehicle comprises 0.5 to 10% co-solvent, 0.5 to 5% co-solvent or 0.5 to 1 % co-solvent. In one embodiment, the reduced co- solvent is DMSO or ethanol.

[0042] The term "a cell" includes a single cell as well as a plurality or population of cells.

[0043] Further provided herein is a pharmaceutical composition comprising 4-amino-5-(4-methylphenyl)-7-(f-butyl)pyrazolo[3,4-of]-pyrimidine ("PP1 ") or (4-amino-5-(4-chlorophenyl)-7-(i-butyl)pyrazolo[3,4-cf]pyrimidine ("PP2") combined with the vehicle disclosed herein.

[0044] Also provided herein is a method of delivering 4-amino-5-(4- methylphenyl)-7-(f-butyl)pyrazolo[3,4-d]-pyrimidine ("PP1") or (4-amino-5-(4- chlorophenyl)-7-(f-butyl)pyrazolo[3,4-d]pyrimidine ("PP2") to a cell or animal in need thereof comprising administering the composition disclosed herein to the cell or animal. In another embodiment, the present disclosure provides use of the composition disclosed herein for delivery of PP1 or PP2 to a cell or animal in need thereof. Also provided herein is use of the composition disclosed herein in the manufacture of a medicament for delivery of PP1 or PP2 to a cell or animal in need thereof. Further provided is a composition disclosed herein for use in delivering PP1 or PP2 to a cell or animal in need thereof. In an embodiment, the methods and uses of delivery are for treating cancer or an acute inflammatory disease or acute inflammatory condition.

[0045] In one embodiment, the compositions, methods and uses disclosed herein comprise PP2. The term "PP2" as used herein refers to a chemical inhibitor of the Src tyrosine kinase family: (4-amino-5-(4- chlorophenyl)-7-(i-butyl)pyrazolo[3,4-d]pyrimidine.

[0046] PP1 is a 1 ^st generation and PP2 is the second. The only difference between them is that PP2 has a [CI] group instead of a [CH3] group

(PP1 ) at the same position on the benzene ring. Both are Src PTK inhibitors.

PP2 is derived from PP1. From the technical sheet, PP2 has lower IC50 for

Src family inhibition than PP1. Accordingly, in another embodiment, the compositions, methods and uses disclosed herein comprise PP1. The term "ΡΡ as used herein refers to a chemical inhibitor of the Src tyrosine kinase family: 4-amino-5-(4-methylphenyl)-7-(f-butyl)pyrazolo[3,4-c]-pyrimidine.

[0047] The compositions described herein can be prepared by per se known methods for the preparation of pharmaceutically acceptable compositions that can be administered to subjects, such that an effective quantity of the active substance is combined in a mixture with a pharmaceutically acceptable vehicle. Suitable vehicles are described, for example, in Remington's Pharmaceutical Sciences (Remington's Pharmaceutical Sciences, 20th ed., Mack Publishing Company, Easton, Pa., USA, 2000). On this basis, the compositions include, albeit not exclusively, solutions of the substances in association with one or more pharmaceutically acceptable vehicles or diluents, and contained in buffered solutions with a suitable pH and iso-osmotic with the physiological fluids.

[0048] Pharmaceutical compositions include, without limitation, lyophilized powders or aqueous or non-aqueous sterile injectable solutions or suspensions, which may further contain antioxidants, buffers, bacteriostats and solutes that render the compositions substantially compatible with the tissues or the blood of an intended recipient. Other components that may be present in such compositions include water, surfactants (such as Tween), alcohols, polyols, glycerin and vegetable oils, for example. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, tablets, or concentrated solutions or suspensions. The pharmaceutical composition may be supplied, for example but not by way of limitation, as a lyophilized powder which is reconstituted with sterile water or saline prior to administration to the patient.

[0049] Pharmaceutical compositions may comprise a pharmaceutically acceptable carrier. Suitable pharmaceutically acceptable carriers include essentially chemically inert and nontoxic compositions that do not interfere with the effectiveness of the biological activity of the pharmaceutical composition. Examples of suitable pharmaceutical carriers include, but are not limited to, water, saline solutions, glycerol solutions, ethanol, N-(1(2,3- dioleyloxy)propyl)N,N,N-trimethylammonium chloride (DOTMA), diolesylphosphotidyl-ethanolamine (DOPE), and liposomes. Such compositions should contain a therapeutically effective amount of the compound, together with a suitable amount of carrier so as to provide the form for direct administration to the patient.

[0050] The composition may be in the form of a pharmaceutically acceptable salt which includes, without limitation, those formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylarnino ethanol, histidine, procaine, etc.

[0051] In accordance with the methods disclosed herein, the compositions disclosed herein may be administered to a subject in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. The compositions may be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump, intratracheal, inhalable or transdermal administration and the pharmaceutical compositions formulated accordingly. Parenteral administration includes intravenous, intravascular, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal and topical modes of administration. Parenteral administration may be by continuous infusion over a selected period of time.

[0052] Accordingly, in one embodiment, the composition is administered by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump, intratracheal, inhalable, intravascular (for example given through an artery such as intracoronary or carotid or given through lymphoid vessels) or transdermal administration. In another embodiment, the formulation is administered by inhalation, intratracheal administration, intravascular administration or intravenous administration. [0053] The compositions disclosed herein may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsules, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the compositions may be incorporated with excipient and used in the form of elixirs, suspensions, syrups, and the like.

[0054] The compositions may also be administered parenterally. Solutions can be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, DMSO and mixtures thereof with or without alcohol, and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. A person skilled in the art would know how to prepare suitable formulations. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2000 - 20th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF 9) published in 1999.

[0055] The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersion and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability exists.

[0056] Compositions for nasal administration or by inhalation may conveniently be formulated as aerosols, drops, gels and powders. Aerosol formulations typically comprise a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent and are usually presented in single or multidose quantities in sterile form in a sealed container, which can take the form of a cartridge or refill for use with an atomising device. Alternatively, the sealed container may be a unitary dispensing device such as a single dose nasal inhaler or an aerosol dispenser fitted with a metering valve which is intended for disposal after use. Where the dosage form comprises an aerosol dispenser, it will contain a propellant which can be a compressed gas such as compressed air or an organic propellant such as fluorochlorohydrocarbon. The aerosol dosage forms can also take the form of a pump-atomizer.

[0057] The compositions may also be delivered by the use of monoclonal antibodies as individual carriers to which the compositions/formulations are coupled. The compositions/formulations may also be coupled with soluble polymers as targetable drug carriers. Such polymers can include polyvinylpyrrolidone, pyran copolymer, polyhydroxypropylmethacrylamide-phenol, polyhydroxy-ethylaspartamide- phenol, or polyethyleneoxide-polylysine substituted with palmitoyl residues. Furthermore, the compositions/formulations may be coupled to a class of biodegradable polymers useful in achieving controlled release of a drug, for example, polylactic acid, polyglycolic acid, copolymers of polyactic and polyglycolic acid, polyepsilon caprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals, polydihydropyrans, polycyanoacrylates and crosslinked or amphipathic block copolymers of hydrogels.

[0058] The dosage of the compositions disclosed herein can vary depending on many factors such as the pharmacodynamic properties of the compound, the mode of administration, the age, health and weight of the recipient, the nature and extent of the symptoms, the frequency of the treatment and the type of concurrent treatment, if any, and the clearance rate of the compound in the animal to be treated. One of skill in the art can determine the appropriate dosage based on the above factors. The compositions/formulations may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response.

[0059] Src family kinases and the downstream STAT pathway have been reported to be involved in cancer development and progression (Gertz, 2008; Hayakawa and Naoe, 2006) and Src and its family of kinase are known as proto-oncogenes (Roskoski, Jr. 2004). In addition, PP2 has been shown to kill thyroid cancer cells (Carlomagno et al., 2003), modulate cell cycle arrest and decrease cervical cancer cell proliferation (Kong et al. 2011 ), decrease the rate of metastasis and retard primary tumour growth of colon cancer as well as increase E-cadherin mediated cell adhesion of colon, liver and breast cancer cells (Nam et al. 2002); decrease cell growth in bladder cancer cell lines (Chiang et al. 2005); decrease glioblastoma cell invasion, proliferation and cell migration; increase growth inhibition of neuroblastoma cells; increase neuroblastoma cell aggregations (Angers-Loustau et al. 2004; Hanke et al. 1996; Hishiki et al. 201 1 ) and decrease the percentage of viable gastrointestinal-neuroendocrine cancer stem cells (Gaur et al. 201 1 ). Further, the present inventors have shown that the disclosed compositions significantly reduced total tyrosine phosphorylation on A549 cells and v-Src transformed NIH-3T3 cells.

[0060] Accordingly, also provided herein is a method of treating cancer comprising administering a pharmaceutical composition comprising 4-amino- 5-(4-methylphenyl)-7-(i-butyl)pyrazolo[3,4-cf]-pyrimidine ("PP1") or (4-amino- 5-(4-chlorophenyl)-7-(i-butyl)pyrazolo[3,4-d]pyrimidine ("PP2") in combination with the vehicle disclosed herein to a subject in need thereof. In another embodiment, the present disclosure provides a use of a pharmaceutical composition comprising PP1 or PP2 in combination with the vehicle disclosed herein for treating cancer. Also provided herein is a use of PP1 or PP2 in combination with the vehicle disclosed herein in the preparation of a medicament for treating cancer. Further provided herein is a pharmaceutical composition comprising PP1 or PP2 in combination with the vehicle disclosed herein for use in treating cancer.

[0061] The term "treating cancer" as used herein refers to reducing or slowing cancerous growth/metastasis or inducing cancer cell death or apoptosis. In one embodiment, the methods and uses for treating cancer comprise administering or using PP1. In another embodiment, the methods and uses for treating cancer comprise administering or using PP2 [0062] The term "cancer" as used herein refers to any kind of cancer of any kind and origin including tumor-forming cells, blood cancers and/or transformed cells. In one embodiment the cancer is thyroid cancer or prostate cancer. In another embodiment, the cancer is cervical cancer, breast cancer, colon cancer, liver cancer, bladder cancer, glioblastoma, neuroblastoma, or gastrointestinal neuroendocrine cancer.

[0063] In yet another embodiment, the present disclosure provides a method of treating acute inflammatory disease or acute inflammatory condition comprising administering a pharmaceutical composition comprising 4-amino-5-(4-methylphenyl)-7-(i-butyl)pyrazolo[3,4-c ]-pyrimidine ("PP1 ") or (4-amino-5-(4-chlorophenyl)-7-(f-butyl)pyrazolo[3,4-c/lpyrimidine ("PP2") in combination with a vehicle disclosed herein to a subject in need thereof. In another embodiment, the present disclosure provides a use of a pharmaceutical composition comprising PP1 or PP2 in combination with a vehicle disclosed herein for treating acute inflammatory disease or acute inflammatory condition. Also provided herein is a use of PP1 or PP2 in combination with a vehicle disclosed herein in the preparation of a medicament for treating acute inflammatory disease or acute inflammatory condition. Further provided herein is a pharmaceutical composition comprising PP1 or PP2 in combination with a vehicle disclosed herein for use in treating acute inflammatory disease or acute inflammatory condition.

[0064] The term "treating acute inflammatory disease or acute inflammatory condition" as used herein refers to countering or inhibiting the inflammatory response, for example, by reducing lymphocyte or neutrophil infiltration and macrophage activation or by reducing pro-inflammatory cytokines, such as TNF-alpha and interleukin 1 beta. In one embodiment, the methods and uses for treating acute inflammatory disease or acute inflammatory condition comprise administering or using PP1. In another embodiment, the methods and uses for treating acute inflammatory disease or acute inflammatory condition comprise administering or using PP2. [0065] The term "acute inflammatory disease or acute inflammatory condition" as used herein refers to any disease or condition resulting in an acute inflammatory response including, without limitation, acute lung injury, brain injury, stroke, myocardial infarction or ischemia-reperfusion induced acute injury in different organ systems.

[0066] The term "treating" or "treatment" as used herein means administering to a subject a therapeutically effective amount of the compositions of the present disclosure and may consist of a single administration, or alternatively comprise a series of applications.

[0067] As used herein, and as well understood in the art, "treatment" or "treating" is also an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results can include, but are not limited to, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. "Treatment" can also mean prolonging survival as compared to expected survival if not receiving treatment. Further any of the treatment methods or uses described herein can be formulated alone or for contemporaneous administration with other agents or therapies. "Treatment" or "treating" can also include preventing the onset of disease.

[0068] The term "subject" as used herein includes all members of the animal kingdom and is optionally a mammal. In one embodiment, the mammal is a human.

[0069] Further provided herein is a method of preparing a formulation of 4-amino-5-(4-methylphenyl)-7-(i-butyl)pyrazolo[3,4-cd-pyrimidine ("PP1") or (4-amino-5-(4-chlorophenyl)-7-(i-butyl)pyrazolo[3,4-c ]pyrimidine ("PP2") with reduced solvent concentration comprising combining the PP1 or PP2 with a vehicle disclosed herein. In one embodiment, the reduced solvent is DMSO. [0070] The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the disclosure. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.

[0071] The following non-limiting examples are illustrative of the present disclosure:

EXAMPLES

Example 1 : The potential of nanoscale combinations of self-assembling peptides and amino acids of the Src tyrosine kinase inhibitor in acute lung injury therapy Results

Problems in PP2 formulation

[0072] Although the Src tyrosine kinase inhibitor PP2 holds therapeutic potentials in the treatments of acute inflammatory responses (Okutani et al., 2006), there exist some challenges to further develop it for clinical uses. The major problem is the very low water solubility of PP2. In previous studies of using PP2 to reduce the ischemia-reperfusion induced acute lung injury in rats, PP2 was attempted to be formulated in saline for intravenous injection. It was found that a minimum of 20% DMSO (by volume) was required to dissolve 0.05 mg/ml PP2 in saline to achieve a therapeutically effective dose of 0.2 mg/kg (Figure 1 A).

[0073] Owing to the well-known toxicity and in vivo side effects of DMSO (in high concentration) (Rubin, 1975), safety tests were conducted by injecting pure DMSO or 20% DMSO in saline to rats via tail or jugular vein (Figure 1 B). It was found that tail vein injection of pure DMSO caused immediate hemolysis and death of the animal; when the amount of DMSO was reduced to 20%, it did not cause acute death of the animal, but hemolysis still occurred leading to blood urine 1 h after injection. On the other hand, no hemolysis was observed when 20%DMSO-saline was slowly injected through the jugular vein. This observed tolerance is probably because of the rapid dilution of DMSO by the relatively higher blood flow in jugular vein than that in tail vein.

[0074] Through this administration route, the PP2-20%DMSO formulation exhibited a protective effect against ischemia-reperfusion induced acute lung injury in rats. However, such a formulation cannot be directly translated into clinical use due to the presence of the high concentration of DMSO. In addition, the reduction of DMSO in the formulation will decrease the PP2 solubility, which ultimately affects the applicable PP2 doses and limits the administration routes for clinical applications. Therefore, new clinically applicable formulation is required in order to develop PP2 into therapeutics.

Nanoscale PP2 formulation: combination of self-assembling peptides with amino acids

[0075] The present formulation strategy for the hydrophobic PP2 was to employ the combination of self-assembling peptides with amino acids to reduce/replace the use of DMSO. The self-assembling peptide EAK16-II was used as a model peptide since it has been found to have good biocompatibility and capability of complexing with hydrophobic therapeutics (Zhang et al., 1995; Fung et al., 2009). The peptide has the hydrophobic side that can interact with hydrophobic molecules (Figure 2); it can also self- assemble into stable nanostructures (Fung et al., 2003), serving as nano- carriers.

[0076] In addition to the self-assembling peptides, single amino acids were introduced into the formulation to replace the use of DMSO. Amino acids bearing similar hydrophobic chemical motifs (e.g., alkyl or aromatic groups) were used to have good compatibility with PP2 (Figure 2), so the PP2 solubility could be enhanced with a much lower amount of DMSO or without DMSO. Furthermore, by introducing amino acids, the osmolarity of the solution can be increased (close to the physiological condition) to avoid hemolytic effects. For such a purpose, 4 amino acids were selected; they either have alkyl side groups (valine V and leucine L) or consist of aromatic structures (phenylalanine F and tryptophan W). The concentrations of these amino acids in the formulation were set to be within their own solubility limits while close to the maximum concentration in clinical uses (as in nutrient infusion solutions). The initial goal was to reduce the use of DMSO to 1 % in the PP2 formulations; eventually, hoping to remove DMSO and replace it with other clinically applicable co-solvents.

[0077] The solubility of PP2 was first tested in 0.1 mg/ml EAK16-II solution with various amounts of DMSO (1-20%). As shown in Figure 3A, the addition of EAK16-II alone did not enhance PP2 solubility even with 20% DMSO as all solutions turned cloudy with precipitation. Similarly, individual amino acid solutions with 1 % DMSO could not solubilize 0.05 mg/ml PP2 (Figure 3B, precipitations at the bottom). The combination of 0.1 mg/ml EAK16-II with the amino acid L, F or W (but not V) was able to formulate 0.05 mg/ml PP2 with the presence of only 1 % DMSO (Figure 3C). The osmolarity of the three formulations were estimated to be about 217 (with L), 214 (with F) and 151 (with W) milli-Osm/L; the former two were close to the physiological value of 308 milli-Osm/L. This was a big achievement as the amount of DMSO in the original formulation was decreased 20 folds with the combination of peptides and amino acids.

[0078] The size of the PP2 nanoformulations was characterized by dynamic light scattering (DLS). All formulations had a size distribution (intensity-based) from 400 nm to 1000 nm with a peak localized around 700 nm.

Hemolytic activity of the PP2 nanoformulations [0079] The next question was if the PP2 nanoformulations are nonhemolytic, which is one critical criterion for most clinical therapeutics developments. The standard ex vivo hemolytic activity assay in pharmaceutical industry was applied. The hemolysis of the formulations can be simply visualized and monitored over 3 h as shown in Figure 4A. None of the three PP2 nanoformulations exhibited significant hemolysis. This was further confirmed by quantitative hemoglobin absorption measurements (Table 2). All tested samples had a hemolysis rate less than 5%, and hence were considered as hemolysis free. For RBC aggregation, it occurred when the EAK16-II concentration was high (0.5 mg/ml); one of the three PP2 nanoformulations (with L) had slight RBC aggregation (Figure 4B). Overall, the PP2 nanoformulations are non-hemolytic.

Src inhibition of PP2 nanoformulations on cultured cells

[0080] Since Src and its family kinases are known as proto-oncogenes (Roskoski, Jr., 2004), two cancer cell lines, A549 and v-Src transformed NIH- 3T3, were employed to test the inhibition of Src activity by the PP2 nanoformulations. PP2 nanoformulations (PP2-F and PP2-L) significantly reduced the total tyrosine phosphorylation on A549 cells in a similar way as the PP2-DMSO control (PP2-D), whereas the vehicle controls (VF and VL) did not have any effect (Figure 5A). Similar results were obtained on the v-Src transformed NIH-3T3 cells (Figure 5B), and the inhibitory effect was pronounced over time (2 h vs. 24 h). In addition, the phosphorylation of STAT3, which is the downstream protein of the Src signaling pathway, was inhibited by the PP2 nanoformulations (Figure 5C). Furthermore, the inhibitory effect can be clearly seen by the change in the cell morphology of the v-Src transformed NIH-3T3 cells upon 24 h treatment with the PP2 nanoformulation as shown in Figure 5D. These results suggest that the nanoformulation does not alter the Src inhibition of PP2 in vitro.

Anti-inflammatory effect of the PP2 nanoformulation in vivo [0081] The therapeutic effect of the PP2 nanoformulation was next evaluated using a simple, widely studied LPS-induced acute lung injury mouse model (Matute-Bello et al., 2008; Wang et al., 2008). The model was illustrated in Figure 6A. The PP2 nanoformulation and the related controls were given intratracheal^ 1 h before the LPS challenging via the same route; the BALF was collected 24 h later to measure inflammatory cell infiltration, total protein concentration and cytokine levels. The PP2 nanoformulation with the amino acid F was tested for its relatively low hemolytic activity (than L, Table 2).

[0082] Prior to the randomized tests, several control experiments were conducted. First, the two intratracheal instillations of saline 1 h apart did not cause any lung inflammation. Second, within the experimental period, the PP2 nanoformulation was found to be safe without any observed adverse responses from the animals. In contrast, the intratracheal administration of the PP2-20%DMSO formulation led to the acute death of mice within 45 min (Figure 6B). The large amount of DMSO in the formulation was thought to destabilize the lung surfactant layer and the cell membranes, leading to the destruction of the lung tissue. This was observed in the majority of the lung (Figure 6C, left), especially for the acute death occurring in less than 10 min. Moreover, it also caused lung inflammation with thicken alveolar walls and cell infiltrations (Figure 6C, right). Third, the LPS dose was optimized to be 1 mg/kg in this model.

[0083] Upon the LPS challenging via air-way, pronounced acute lung inflammation was observed (Figure 7): the total cell number was dramatically increased in the BALF with severe neutrophils infiltration (Figure 7A and B); the total protein concentration in the BALF was significantly increased (Figure 7C), and so was the pro-inflammatory cytokine TNF-ot (Figure 7D). The pretreatment of the PP2 nanoformulation was found to be able to significantly reduce the LPS-induced cell infiltration and TNF-ot production in the lung (Figure 7A, B and D). However, the decrease in the total protein concentration upon the pretreatment of the PP2 nanoformulation was not significant (Figure 7C). Both LPS and PP2 treatment did not affect the regulatory cytokine IL-10 level in the BALF (Figure 7E). These results demonstrated that the PP2 nanoformulation had anti-inflammatory effects on the LPS-induced acute lung injury by reducing the inflammatory cell infiltration and TNF-a production.

Discussion

The use of DMSO in drug formulation

[0084] DMSO is widely accepted and used as a common solvent for many hydrophobic bioactive agents in laboratory experiments. However, such a solvent (in high amount) is cytotoxic and harmful in vivo, and not allowed to be used in most pharmaceutical products; the only clinical uses approved by Food and Drug Administration (FDA) are in tissue preservation for bone marrow transplantation and as treatments for interstitial cystitis (Rubin, 1975; Sauer-Heilborn et al., 2004).

[0085] Despite that DMSO has shown therapeutic evidence in treating pain, inflammation, scleroderma, interstitial cystitis and arthritis elevated intracranial pressure, its toxicity is well-documented (Santos et al., 2003; Hameroff et al., 1983; Rubin, 1983). Several side effects have been described during cutaneous application or intravenous administration of DMSO (Sauer- Heilborn et al., 2004); the common symptoms include sedation, headache, nausea and dizziness. DMSO is also known to induce histamine release, causing hypotension or anaphylactic infusion reactions. Intravascular hemolysis, hyperosmolality, and increased serum transaminase levels have been reported after its intravenous administration in humans.

[0086] Indeed, the present experimental data also shows that large amounts of DMSO (20% or above) in the PP2 formulations is toxic in a rat or mouse model. It can cause hemolysis and acute death of animals (in severe case) when intravenously administered (Figure 1 B); the intratracheal instillation of 20% DMSO will also lead to acute animal death (Figure 6B). This may be related to the propensity of DMSO in the modulation of membrane structure and stability (Yu and Quinn, 1998).

[0087] By using the combination of peptides and amino acids in PP2 formulation, the use of DMSO was able to be significantly reduced from 20% to 1 %. The presence of 1 % DMSO is within the FDA approval range for the clinical use in bone marrow transplantation (Sauer-Heilborn et al., 2004). In addition, the PP2 nanoformulations did not exhibit any hemolytic activity and severe side effect in acute phase in vivo. The vehicle alone (without PP2) also showed no trace of acute toxicity. This suggests that the present nanoformulations are relatively safe and non-toxic regardless of the presence of 1 % DMSO. To further improve such formulations, it is desirable to completely replace DMSO with other clinically applicable co-solvents, such as ethanol, or other non-toxic excipients. Achievements of the nanoformulations toward clinical applications

[0088] The present inventors have developed a nanoscale formulation strategy, using the combination of self-assembling peptides and amino acids, to improve the biocompatibility of the previous PP2 formulation for potential clinical use.

[0089] Such a formulation has accomplished the following advances. First, the use of the toxic organic co-solvent (DMSO) can be significantly reduced (20-fold). Second, adding amino acids can increase the osmotic pressure of the formulation and decrease the use of peptides, which minimizes the immunogenic potential. Third, the formulations are proven to be non-hemolytic and safe for intravenous and intratracheal administration. Fourth, the formulations do not alter the biological activity (Src inhibition) of PP2 in vitro. Fifth, they show anti-inflammatory effect on LPS-induced pulmonary inflammation in mice. The single dose of PP2 given in the animal model is 0.2 mg/kg, which is 5 times lower than that used in similar models (Severgnini et al., 2005). [0090] One potential clinical application is to treat acute inflammatory response related diseases. Many reports have shown that PP1/PP2 can reduce acute inflammation in acute lung injury, brain injury, spinal cord compression, stroke and myocardial infarction on various animal models (Okutani et al., 2006; Khadaroo et al., 2004; Severgnini et al., 2005; Lee et al., 2007). Thus, the developed PP2 nanoformulations in this study would help translate its use in these clinical settings. In addition, the recent positive results of PP2 as treatments for the ischemia-reperfusion induced ALI on a rat model suggest its potential clinical use as the anti-inflammatory therapeutics for lung transplantation. Another application could be anticancer therapy. Src family kinases and the downstream STAT pathway has been reported to be involved in cancer development and progression (Gertz, 2008; Hayakawa and Naoe, 2006). Thus, inhibition of Src pathway by the PP2 nanoformulations (Figure 5) raises their implications in anticancer treatments. Materials and Methods

Self-assembling peptides

[0091] The self-assembling peptide EAK16-II (1657.66 g/mol, > 95% pure by HPLC) was purchased from CanPeptide Inc. (Pointe-Claire, Canada). The amino acid sequence of EAK16-II is n-AEAEAKAKAEAEAKAK-c (SEQ ID NO:2), where A corresponds to alanine, E to glutamic acid and K to lysine. The N-terminus and C-terminus of the peptide were protected by acetyl and amino groups, respectively.

Reagents and antibodies

[0092] All amino acids (reagent grade, > 98% pure), the organic solvent dimethyl sulfoxide (DMSO, > 99% pure) and the endotoxin lipopolysaccharide (LPS, from Escherichia coli 055:B5) were obtained from Sigma-Aldrich (Oakville, Canada). Dulbecco's modified eagle medium (DMEM) and phosphate buffer saline (PBS) were from our Tissue Culture Media Facility (University Health Network, Toronto, Canada), while fetal bovine serum (FBS) were from Invitrogen Inc. (Burlington, Canada). Other chemicals were either from Sigma-Aldrich (Oakville, Canada) or from BDH Inc. (Toronto, Canada). The antibodies against total tyrosine phosphorylation (4G10), total and phosphorylated STAT3 were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Anti-GAPDH antibody was from Upstate (Billerica, MA). Horseradish peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit secondary antibodies were acquired from Amersham Pharmacia Biotech (Piscataway, NJ). The Src tyrosine kinase inhibitor PP2 (> 98% pure) was purchased from BIOMOL International (Plymouth Meeting, PA). PP2 formulation

[0093] The original PP2 formulations for intravenous injection were made of 0.05 mg/ml PP2 in a standard physiological saline solution containing various amounts of DMSO (5-25% by volume).

[0094] The PP2 nanoformulation was made by diluting PP2-DMSO stock (5 mg/ml) into peptide solutions alone, amino acid solutions alone or peptide-amino acid mixtures to have a final PP2 concentration of 0.05 mg/ml. The PP2-DMSO stock solution was prepared by dissolving 1 mg PP2 in pure DMSO and stored in -20 °C prior to further dilution. The peptide solution was freshly prepared in pure water (Milli-Q, 18.2 ΜΩ), and so were the amino acid solutions, whereas the peptide-amino acid mixtures were made by dissolving peptide powders in the amino acid solutions. These solutions were subsequently under bath sonication for 10 min to completely dissolve peptides. They were then mixed with PP2-DMSO stock vigorously (on a Vortex Mixture), and the final PP2 nanoformulations would consist of different percentages (by volume) of DMSO from 1 to 20%. The formulations were centrifuged at 13,200 rpm for 1 min to observe any precipitations; those without visible precipitations were considered as "soluble" PP2 nanoformulations and were further tested.

Dynamic light scattering (DLS) [0095] The hydrodynamic diameter of the PP2 nanoformulations was obtained using Zetasizer Nano Z90 (Malvern Instruments, Worcestershire, U.K.). Formulated solutions (1 ml) were placed in a disposable plastic cuvette. The temperature was maintained at 25 °C and the solution was equilibrated for 2 min before data acquisition. Four measurements were performed for each sample. The data was automatically analyzed with the software to generate the intensity-based size distribution of the formulations.

Hemolytic activity test

[0096] To evaluate the in vivo toxicity of the PP2-DMSO formulation, pure DMSO (1 ml/kg) and 20% DMSO in saline (4 ml/kg) was injected to male Sprague Dawley rat (250-300 g) via either tail vein or jugular vein. The rats were monitored continuously for their acute reaction; their blood and urine were then collected immediately or 1 h after injection to examine potential hemolysis. All animal studies herein followed the protocols approved by the Animal Care Committee at the University Health Network - Toronto General Research Institute.

[0097] The hemolytic activity of the "soluble" PP2 nanoformulations was studied ex vivo using fresh pig blood (Yorkshire male domestic pigs, 25- 35 kg). The assay followed the standard pharmaceutical industry procedures. In brief, the pig blood was freshly collected and under vigorously stirring by a glass stick to remove fibrinogen. It was washed 3-5 times with a standard physiological saline solution to preserve intact red blood cells (RBC) and diluted to 2% (by volume) in saline. Test samples were diluted and mixed with RBC suspensions so the final mixtures (5 ml) contained 1 % RBC and various concentrations of the samples. For each sample, 5 different dilutions (5-50 fold) were tested; the preparation for each dilution was according to Table 1. Pure water and standard physiological saline solution were used as the positive and negative control, respectively. The mixtures were incubated in water bath at 37 °C to allow the RBC precipitation, and the color of the upper solution was monitored every 30 min for 3 h. At 3 h, the mixtures were centrifuged at 1 ,500 rpm for 10 min; the supernatants were transferred to a 96-well plate, which was read by a microplate reader (Opsys MR, Thermo Labsystems) for absorption at 595 nm. The sample absorbance was compared with that of positive and negative control samples to determine Hemolysis % by the following equation:

Absfsamples)— Abs—

Hemolysis % = X 100*

[0098] Briefly, after cells were harvested and disrupted in lysis buffer, the extracted proteins in the lysate supernatant were first analyzed with a modified Bradford assay (Bio-Rad, Munich, Germany) to determine the total protein concentration; hence the protein concentration in the lysates were adjusted accordingly prior to the SDS-PAGE (10%) gel separation process. The separated proteins were then transferred from the gel onto nitrocellulose membranes (Schleicher & Schuell, Whatman, Middlesex, UK). The membranes were blotted with different primary antibodies and secondary antibodies coupled with horseradish peroxidase. Targeted proteins were revealed by an enhanced chemiluminescence detection kit (SuperSignal West Dura Substrate, Thermo Scientific, Rockford, IL) followed by the film development.

LPS-induced acute lung injury mouse model

[0099] Male Balb/c mice (6-8 weeks, 22-28 g) were used to establish the LPS-induced acute lung injury model (Han et al., 2010; He et al., 2010). LPS (1 mg/kg in saline) was given intratracheally by direct injection through the orotracheal route with a surgical microscope (Leica M651 , Richmond Hill, Canada). After 24 h, mice were euthanized and the bronchoalveolar lavage fluid (BALF) was collected for lung inflammation assessments. [00100] The PP2 nanoformulation (0.2 mg/kg) and standard physiological saline solution were given 1 h before the LPS challenging through the same administration route to evaluate its anti-inflammatory effect. Mice were randomized and the treatments were given in blinded fashion. Each group contained 11 mice, while 5-6 mice were used for the negative and vehicle control groups. Mice were under anesthesia (Ketamine/Xylazine cocktail, 140 mg/kg and 7 mg/kg, respectively, ip) during the two injections and kept in a MediHEAT recovery chamber (Peco Services Ltd., Cumbria, UK) to maintain their body temperature.

[00101] Vehicle control was tested the same way without LPS challenging to examine the acute toxicity of the formulation materials. In the meanwhile, the original formulation of PP2 in saline with 20% DMSO was also given intratracheally to a total of 10 mice for comparison.

BALF collection and lung inflammation assessments

[00102] BAL was performed based on the procedure described previously (Mura et al., 2006). In brief, the tracheal intubation was first established on the euthanized mice. Iced cold PBS (1 ml) was injected through the endotracheal tube and gently aspirated back. A total of 3 ml PBS was instilled and -80% of the fluid was recovered. An aliquot of BALF (50 μΙ) was transferred to a centrifuge tube and mixed with equal volume trypan blue solution for total cell counting using a hemocytometer. In parallel, an aliquot of BALF (80 μΙ) underwent cytospin (Shandon CytoSpin II) at 800 rpm for 5 min; the cells collected on the glass slide were stained using the Harleco Hemacolor staining kit (EMD Science, Gibbstown, NJ, USA) and imaged (200x) under a microscope (Nikon Eclipse 80i). Differential cell count was conducted on 40-50 images/sample. The remainder of the BALF was centrifuged at 1 ,500 rpm, 4 °C for 10 min, and the supernatant was stored at - 80 °C until the measurement of the total protein concentration by the modified Bradford assay (Bio-Rad Laboratories, Hercules, CA, USA) and cytokine quantification with Enzyme-Linked Immunosorbent Assay (ELISA). [00103] The pro-inflammatory cytokine TNF-ot and the regulatory cytokine IL-10 in the BALF were quantified using mouse DuoSet ELISA kits (R&D Systems, Minneapolis, MN). The assay was performed following the manufacture's instruction. Lung histology

[00104] Lungs were dissected and inflated at a 20 cm height with 4% paraformaldehyde in PBS for fixation for 24 h. They were further processed in a tissue processor (Excelsior ES, Thermo Scientific) prior to be embedded in paraffin. Tissue section (5 μιτι) was performed and fixed on the glass slide, followed by haematoxylin and eosin staining. Stained sections were imaged under an optical microscope (Nikon Eclipse 80i) at different magnifications.

Statistical analysis

[00105] Statistical analysis was carried out using ANOVA or Student's t- test where applicable. In vitro experiments were repeated at least three times. Differences were considered significant when the P value was less than 0.05.

Example 2: PP2 Nanoformulations with Various Amino Acids

[00106] Table 3 shows the results of nanoformulations produced with 19 amino acids. Cysteine was not tested because it is seldom used clinically and it can form S-S bonds. Using a solubility-improvement threshold of 2 or more solubility steps as quantified (low cone. AA and high cone. AA) in the table between "no precipitation" (denoted as "x") and "very high precipitation", leucine, phenylalanine, tryptophan, or methionine provide the best solubility at the given concentration for each amino acid. A lower threshold of just 1 solubility step shows alanine, isoleucine and asparagine also provide solubility at the given concentration for each amino acid. Glutamic and aspartic acid may lower the solution pH, so PP2 might be solubilized by protonation (no pellets have been observed in any of the experiments). The preparation method of these formulations is the same as described in Example 1. Example 3: Nanoformulations with Self-assembling Peptides

[00107] Different self-assembling peptides are employed in addition to EAK16-II. The peptides are listed in Table 4. EAK16-I and EAK16-IV have different charge distribution than EAK16-II. The formulation method is the same as described in Example 1. Those formulations that can solubilize the PP1 or PP2 are examined on cultured cells to confirm their therapeutic functions.

[00108] While the present disclosure has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the disclosure is not limited to the disclosed examples. To the contrary, the disclosure is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.

[00109] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety.

Table 1 Sample dilution for hemolytic activity assay

Labels 2% RBC (ml) Saline (ml) Pure H₂0 Samples (ml)

(ml)

1 2.5 2.4 0 0.1

2 2.5 2.3 0 0.2

3 2.5 2.2 0 0.3

4 2.5 2.1 0 0.4

5 2.5 2.0 0 0.5

+ 2.5 0 2.5 0

- 2.5 2.5 0 0

Table 2 Hemolytic activity of EAK16-II alone and the PP2 nanoformulations

Table 3

Dissolvin PP2

PBS stock solution

Table 4

N- and C-termini are protected via acetylation and amidation, respectively.

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Claims

CLAIMS:

1. A vehicle for delivering 4-amino-5-(4-methylphenyl)-7-(i-butyl)pyrazolo[3,4- ci]-pyrimidine ("PP1") or (4-amino-5-(4-chlorophenyl)-7-(f-butyl)pyrazolo[3,4- cf]pyrimidine ("PP2") comprising a self-assembling peptide in combination with a single amino acid, wherein the self-assembling peptide consists of lysine (K), glutamate (E) and alanine (A) amino acid residues.

2. The vehicle of claim 1 , wherein the single amino acid comprises leucine, phenylalanine, tryptophan, or methionine.

3. The vehicle of claim 1 , wherein the single amino acid comprises alanine, isoleucine or asparagine.

4. The vehicle of claim 1 , wherein the single amino acid comprises glutamic acid or aspartic acid.

5. The vehicle of any one of claims 1-4, wherein the self-assembling peptide comprises 12-30 amino acid residues. 6. The vehicle of claim 5, wherein the self-assembling peptide comprises 6 amino acid residues.

7. The vehicle of any one of claims 1 to 6, wherein the self-assembling peptide is EAK16-I, EAK16-II or EAK16-IV.

8. The vehicle of claim 7, wherein the self-assembling peptide is EAK16-II. 9. The vehicle of any one of claims 1-8, further comprising 0.5 to 10% co- solvent.

10. The vehicle of claim 9, comprising 0.5 to 5% co-solvent.

11. The vehicle of claim 10, comprising 0.5 to 1% co-solvent.

12. The vehicle of any one of claims 9-11, wherein the co-solvent is DMSO or ethanol.

13. A pharmaceutical composition comprising 4-amino-5-(4-methylphenyl)-7- (f-butyl)pyrazolo[3,4-<¾-pyrimidine ("PP1") or (4-amino-5-(4-chlorophenyl)-7-(f- butyl)pyrazolo[3,4-c/]pyrimidine ("PP2") combined with the vehicle of any one of claims 1-12.

14. The pharmaceutical composition of claim 13, further comprising pharmaceutically acceptable vehicles and/or diluents. 5. The pharmaceutical composition of claim 13 or 14 formulated as an inhalant, for intravenous administration or for intratracheal administration.

16. The pharmaceutical composition of any one of claims 13 to 15, comprising PP2.

17. The pharmaceutical composition of any one of claims 13 to 15, comprising PP1. 18. A use of the pharmaceutical composition of any one of claims 13 to 17 for delivering 4-amino-5-(4-methylphenyl)-7-(i-butyl)pyrazolo[3,4-d]-pyrimidine ("PP1 ") or (4-amino-5-(4-chlorophenyl)-7-(f-butyl)pyrazolo[3,4-d]pyrimidine ("PP2") to a cell or animal in need thereof.

19. The use of claim 18, wherein the composition is used by inhalation, intratracheally or intravenously.

20. A use of 4-amino-5-(4-methylphenyl)-7-(f-butyl)pyrazolo[3,4-cf]-pyrimidine ("PP1") or (4-amino-5-(4-chlorophenyl)-7-(f-butyl)pyrazolo[3,4-o]pyrimidine ("PP2") in combination with the vehicle of any one of claims 1-12 for treating cancer in a subject in need thereof. 21. A use of a pharmaceutical composition comprising 4-amino-5-(4- methylphenyl)-7-(i-butyl)pyrazolo[3,4-o]-pyrimidine ("PP1") or (4-amino-5-(4- chlorophenyl)-7-(i-butyl)pyrazolo[3,4-c]pyrimidine ("PP2") in combination with the vehicle of any one of claims 1-12 for treating acute inflammatory disease or acute inflammatory condition in a subject in need thereof.

22. A method of preparing a pharmaceutical composition of 4-amino-5-(4- methylphenyl)-7-(i-butyl)pyrazolo[3,4-c]-pyrimidine ("PP1") or (4-amino-5-(4- chlorophenyl)-7-(f-butyl)pyrazolo[3,4-c/]pyrimidine ("PP2") with reduced solvent concentration comprising combining PP1 or PP2 with the vehicle of any one of claims 1-12.

23. The method of claim 22, wherein the reduced solvent is DMSO or ethanol.