Nanocomplexes of modified peptides or proteins

Abstract

A nanocomplex containing a delivery agent and a pharmaceutical agent. The nanocomplex has a particle size of 50 to 1000 nm, the delivery agent binds to the pharmaceutical agent via non-covalent interaction or covalent bonding, and the pharmaceutical agent is a modified peptide or protein formed of a peptide or protein and an added chemical moiety that contains an anionic group, a disulfide group, a hydrophobic group, a pH responsive group, a light responsive group, a reactive oxygen species responsive group, or a combination thereof.

Claims

1. A nanocomplex comprising a delivery agent and a pharmaceutical agent, wherein the nanocomplex has a particle size of 50 to 1000 nm, the delivery agent binds to the pharmaceutical agent via non-covalent interaction or covalent bonding, and the pharmaceutical agent is a modified peptide or protein, comprising a peptide or protein and an added chemical moiety, wherein the added chemical moiety comprises a reactive oxygen species responsive group, wherein the reactive oxygen species responsive group comprises a boronic ester group; and the added chemical moiety is: ##STR00027## wherein: W is the boronic ester group; X is O, S, or NR.sub.e, wherein R.sub.e, is H, OH, an amino acid radical, a peptide radical, a C.sub.1-C.sub.10 monovalent aliphatic radical, a C.sub.1-C.sub.10 monovalent heteroaliphatic radical, a monovalent aryl radical, or a monovalent heteroaryl radical; Y is a bond, O, S, or NR.sub.f, wherein R.sub.f is H, OH, an amino acid radical, a peptide radical, a C.sub.1-C.sub.10 monovalent aliphatic radical, a C.sub.1-C.sub.10 monovalent heteroaliphatic radical, a monovalent aryl radical, or a monovalent heteroaryl radical; and Z is R.sub.h-A.sub.7-A.sub.8-R.sub.i, wherein R.sub.h and R.sub.i, independently, are a bond or a C.sub.1 aliphatic radical; A.sub.7 is a bivalent aryl radical or a bivalent heteroaryl radical; and A.sub.8 is a bond; and the delivery agent is a surfactant, a lipid, a lipid-like compound, a polymer, a dendrimer, an inorganic nanoparticle, a polymer nanoparticle, an inorganic nanowire, a polymer nanowire, a nanotube, or a combination thereof; wherein the lipid-like compound has the following formula: ##STR00028## wherein: V is a hydrophilic head: E is a C.sub.1-C.sub.24 monovalent aliphatic radical, a C.sub.1-C.sub.24 monovalent heteroaliphatic radical, a monovalent aryl radical, a monovalent heteroaryl radical, or T.sub.4-U.sub.2-T.sub.5-SS-T.sub.6; T.sub.2-SS-T.sub.3 is a hydrophobic tail; and U.sub.1 and U.sub.2 are each a linker, wherein each of T.sub.1 and T.sub.4, independently, is a bond, a C.sub.1-C.sub.10 bivalent aliphatic radical, a C.sub.1-C.sub.10 bivalent heteroaliphatic radical, a bivalent aryl radical, or a bivalent heteroaryl radical; each of T.sub.2 and T.sub.5, independently, is a bond, a C.sub.1-C.sub.20 bivalent aliphatic radical, a C.sub.1-C.sub.20 bivalent heteroaliphatic radical, a bivalent aryl radical, or a bivalent heteroaryl radical; each of T.sub.3 and T.sub.6, independently, is a C.sub.1-C.sub.20 monovalent aliphatic radical, a C.sub.1-C.sub.20 monovalent heteroaliphatic radical, a monovalent aryl radical, or a monovalent heteroaryl radical; and SS stands for a disulfide moiety.

2. The nanocomplex of claim 1, wherein the added chemical moiety is linked to the peptide or protein via an amide group, an ester group, an amidine group, a urea group, a carbamate group, an imidoester group, or a carbonate group.

3. The nanocomplex of claim 2, wherein the added chemical moiety is linked to the peptide or protein via an amide group, an ester group, or a carbamate group.

4. The nanocomplex of claim 1, wherein the added chemical moiety is: ##STR00029##

5. The nanocomplex of claim 1, wherein the delivery agent is a surfactant, a lipid, a lipid-like compound, a polymer, or a combination thereof.

Description

DETAILED DESCRIPTION

(1) The nanocomplex of this invention contains a modified peptide or protein. The modification can be achieved by attaching to a peptide or protein, non-covalently or covalently, a chemical moiety that contains an anionic group, a disulfide group, a hydrophobic group, a pH responsive group, a light responsive group, a reactive oxygen species responsive group, or a combination thereof.

(2) Non-covalent bonding refers to ionic interaction, hydrogen bonding, van der Waals interaction, and hydrophobic interaction. On the other hand, covalent bonding is typical formed by crosslinking a chemical moiety to a functional group (e.g., NH.sub.2, NH, COOH, SH, C(O), and CHO) on a peptide or protein.

(3) Crosslinking agents are generally used to modify a peptide or protein. Examples include anhydrides, carbonates, sulfonothinates, carbodiimides, disuccinimidyl suberate, symmetric disulfides, sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate, N-hydroxysuccinimide esters, imidoesters, maleimides, haloacetyls, alkyl halides, pyridyl disulfides, hydrazides, alkoxyamines, aryl azides, and diazirines. The exemplary crosslinking agents are described in detail below.

(4) An anhydride reacts with a NH.sub.2 or NH group on a peptide or protein. Cyclic anhydrides and anionic group-containing anhydrides are particularly useful. Examples include cis-aconitic anhydride, 4-methyl-2,5-dioxo-2,5-dihydrofuran-3-carboxylic acid, 2,6-dioxo-5,6-dihydro-2H-pyran-3-carboxylic acid, and 4-methyl-2,6-dioxo-5,6-dihydro-2H-pyran-3-carboxylic acid.

(5) A carbonate also reacts with NH.sub.2/NH to form a carbamate group. Examples include

(6) ##STR00005##

(7) A sulfonothinate (e.g., 3-(methylsulfonylthio)propanoic acid) reacts with the SH group on a cysteine residue in a peptide or protein, forming a disulfide bond.

(8) A carbodiimide, e.g., N,N-dicyclohexylcarbodiimide and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide), activates a carboxyl group or a phosphate group to couple it with NH.sub.2/NH to yield an amide bond.

(9) Disuccinimidyl suberate has the following formula:

(10) ##STR00006##
It reacts with NH.sub.2/NH to form an amide group.

(11) A symmetric disulfide has the following formula: RSSR, in which R contains an anionic group. It reacts with SH to form a mixed disulfide group. Examples include 5,5-dithio-bis-(2-nitrobenzoic acid) and N,N-didansyl-L-cystine.

(12) Sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate has the following formula:

(13) ##STR00007##
It can react with NH.sub.2/NH to form an amide group and can also react with SH to form a thioether group.

(14) An N-hydroxysuccinimide ester has the following formula:

(15) ##STR00008##
It reacts with NH.sub.2/NH to form an amide group.

(16) An imidoester has the following formula:

(17) ##STR00009##
It reacts with NH.sub.2/NH to form an amidine group.

(18) A maleimide has the following formula:

(19) ##STR00010##
It reacts with SH to form a thioether group.

(20) A haloacetyl has the following formula:

(21) ##STR00011##
in which Hal is I or Br. It can react with NH.sub.2/NH to form an amine group and can also react with SH to form a thioether group.

(22) An alkyl halide has the following formula: RCH.sub.2Hal. It can react with NH.sub.2/NH to form an amine group and can also react with SH to form a thioether group.

(23) A pyridyl disulfide has the following formula:

(24) ##STR00012##
It reacts with SH to form a disulfide group.

(25) A hydrazide has the following formula:

(26) ##STR00013##
It reacts with a carbonyl group (aldehyde and ketone) in a peptide or protein to form a hydrazone group.

(27) An alkoxyamine has the following formula:

(28) ##STR00014##
It reacts with a carbonyl group to form an imidoester group.

(29) An aryl azide has the following formula:

(30) ##STR00015##
Upon being exposed to UV light, the aryl group insert into CH or NH on a peptide or protein.

(31) A diazirine has the following formula:

(32) ##STR00016##
Like an aryl azide, it insert into CH or NH upon being exposed to UV light.

(33) A peptide or protein can react with 4-chloro-7-nitro-1,2,3-benzoxadiazole to form a amine or thioether group and can also react with methyl methanethiosulfonate to form SSCH.sub.3.

(34) In addition, a peptide or protein can be modified through a traceless Staudinger ligation. See Bernardes et al., ChemBioChem, 12, 1383-86 (2011); Tam et al., Methods Enzymol., 462, 25-44 (2009); and Hayashi et al., Accounts of Chemical Research, 45, 1460-69 (2012).

(35) More examples of peptide/protein modification are described in Lee et al., Angewandte Chemie International Edition, 48, 5309-12 (2009); Lee et al., Angewandte Chemie International Edition, 49, 2552-55 (2010); and Maier et al., Journal of American Chemical Society, 134, 10169-73 (2012).

(36) Preferably, the peptide/protein modification is reversible. Namely, after the modified peptide/protein, as a component of a nanocomplex, enters a cell, the added chemical moiety can be cleaved by a redox enzyme or light, or as a result of pH change.

(37) For example, a protein containing a lysine residue is modified with a disulfide moiety. After the modified protein enters a cell, the disulfide moiety is removed by glutathione (hereinafter GSH) or other cysteine residues to regenerate the nascent protein. See Scheme 1 below.

(38) ##STR00017##

(39) As another example, a protein containing a cysteine residue is modified with a negatively charged disulfide moiety. After the modified protein enters a cell, the moiety is then reduced by GSH, as shown in Scheme 2 below:

(40) ##STR00018##

(41) As a further example, a protein is modified with a light responsive moiety. After being delivered to a cell, the modified protein is exposed to light to remove the moiety. See Scheme 3 below.

(42) ##STR00019##

(43) As still another example, a protein is modified with a pH responsive moiety. After the modified protein enters a cell, the moiety is cleaved in, e.g., an endosome of the cell. See Scheme 4 below.

(44) ##STR00020##

(45) As yet another example, a protein is modified with a reactive oxygen species (ROS) responsive moiety, shown in Scheme 5 below as BNC. After the modified protein enters a cell, the moiety is cleaved by ROS, e.g., H.sub.2O.sub.2. See Scheme 5.

(46) ##STR00021##

(47) The peptide/protein thus modified has an isoelectric point of 9.6 to 3.6 (e.g., 8.5 to 4.8 and 6.5 to 5.5). Nanocomplexes containing the modified peptide/protein described above are useful for delivering a peptide or protein into a cell. These nanocomplexes can be preliminarily screened for their delivery efficacy by an in vitro assay and then confirmed by animal studies and clinic trials. Other methods will also be apparent to those of ordinary skill in the art.

(48) The above-described nanocomplexes can be prepared using the procedures described in publications such as Wang et al., ACS Synthetic Biology, 1, 403-07 (2012); Kim et al., Biomaterials, 33, 3959-66 (2012); Gonzalez-Toro et al., Journal of American Chemical Society, 134, 6964-67 (2012); and Slowing et al., Journal of American Chemical Society, 129, 8845-49 (2007). Generally, they are obtained by incubating a lipid-like compound and a pharmaceutical agent in a buffer such as a sodium acetate buffer or a phosphate buffer. The nanocomplexes thus obtained have a particle size of 50 to 1000 nm (e.g., 50 to 500 nm, 50 to 300 nm, and 50 to 180 nm).

(49) Further, this invention covers a pharmaceutical composition containing the nanocomplex described above and a pharmaceutically acceptable carrier. The pharmaceutical carrier is compatible with the nanocomplex and should not be deleterious to the subject to be treated.

(50) Moreover, another aspect of this invention relates to a method of administering an effective amount of the nanocomplex described above to a patient in need. An effective amount refers to the amount of the nanocomplex that is required to confer a therapeutic effect on the treated subject. Effective doses will vary, as recognized by those skilled in the art, depending on the types of diseases treated, route of administration, excipient usage, and the possibility of co-usage with other therapeutic treatment.

(51) To practice the method of the present invention, a composition having the above-described nanocomplex can be administered parenterally, orally, nasally, rectally, topically, or buccally. The term parenteral as used herein refers to subcutaneous, intracutaneous, intravenous, intramuscular, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, or intracranial injection, as well as any suitable infusion technique.

(52) A sterile injectable composition can be a solution or suspension in a non-toxic parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that can be employed are mannitol, water, Ringer's solution, and isotonic sodium chloride solution. In addition, fixed oils are conventionally employed as a solvent or suspending medium (e.g., synthetic mono- or diglycerides). Fatty acid, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharmaceutically acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions can also contain a long chain alcohol diluent or dispersant, carboxymethyl cellulose, or similar dispersing agents. Other commonly used surfactants such as Tweens or Spans or other similar emulsifying agents or bioavailability enhancers which are commonly used in the manufacture of pharmaceutically acceptable solid, liquid, or other dosage forms can also be used for the purpose of formulation.

(53) A composition for oral administration can be any orally acceptable dosage form including capsules, tablets, emulsions and aqueous suspensions, dispersions, and solutions. In the case of tablets, commonly used carriers include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions or emulsions are administered orally, the active ingredient can be suspended or dissolved in an oily phase combined with emulsifying or suspending agents. If desired, certain sweetening, flavoring, or coloring agents can be added.

(54) A nasal aerosol or inhalation composition can be prepared according to techniques well known in the art of pharmaceutical formulation. For example, such a composition can be prepared as a solution in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other solubilizing or dispersing agents known in the art.

(55) A composition having the nanocomplexes can also be administered in the form of suppositories for rectal administration.

(56) The specific examples below are to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. Without further elaboration, it is believed that one skilled in the art can, based on the description herein, utilize the present invention to its fullest extent. All publications cited herein are hereby incorporated by reference in their entirety.

Example 1

Preparation of Lipid-Like Compound EC16-63

(57) Compound EC16-63 was prepared using N,N-dimethyl-1,3-propanediamine and 1,2-epoxyhexadecane, as shown below:

(58) ##STR00022##

(59) In a 5-mL Teflon-lined glass screw-top vial, 1,2-epoxyhexadecane was added to N,N-dimethyl-1,3-propanediamine at a molar ratio of 2.4:1. The mixture was stirred at 80 C. for two days. After cooling, lipid-like compound EC16-63 thus formed was purified using flash chromatography on silica gel and characterized by proton nuclear magnetic resonance.

(60) Protein Modification

(61) Protein RNase A was modified using cis-aconitic anhydride following the procedures described below.

(62) RNase A was dissolved in 0.1 M NaHCO.sub.3 buffer solution (pH=9.5). After the resultant solution was cooled to 4 C., cis-aconitic anhydride was added in two batches, i.e., 50 mg and 10 mg. After stirring at room temperature for two hours, the reaction was complete to yield the aconitic acid modified RNase A (hereinafter RNase A-Aco), which was enriched by centrifugal filtration three times (Amicon Ultra, MWCO=10000, Millipore, Billerica, Mass.) and subsequent dialysis against NaHCO.sub.3 buffer (25 mM, pH=9.5) for 24 hours.

(63) Preparation of Composition A

(64) Compound EC16-63 was dissolved in a phosphate buffer (25 mM, pH=7.4) at a concentration of 12 g/mL. RNase A-Aco was added to the resultant mixture and incubated for 15 minutes at room temperature. The weight ratio between Compound EC16-63 and RNase A-Aco was 6:5.

(65) Comparative Composition A was also prepared following the same procedure described above except that RNase A was used instead of RNase A-Aco.

(66) Both compositions were subjected to the in vitro assay described in Example 5 below. The results are also shown in that example.

Example 2

(67) Composition B was prepared following the same procedure described in Example 1 above except that a light responsive RNase A was used instead of RNase A-Aco.

(68) The light responsive RNase A was obtained following the same procedure also described in Example 1 except that carbonate

(69) ##STR00023##
was used instead of cis-aconitic anhydride. See Scheme 3 above.

(70) Comparative Composition B was prepared following the same procedure used to prepare Composition B, except that no lipid-like compound was used.

(71) Both compositions were subjected to the in vitro assay described in Example 5 below and the results are shown in FIG. 1 also below.

Example 3

(72) Composition C was prepared using the same procedure described in Example 1 above except that a disulfide modified RNase A was used instead of RNase A-Aco.

(73) The disulfide modified RNase A was obtained following the same procedure described also in Example 1 except that disulfide

(74) ##STR00024##
was used instead of cis-aconitic anhydride. See Scheme 1 above.

(75) Comparative Composition C was prepared following the same procedure used to prepare Composition C, except that no lipid-like compound was used.

(76) The two compositions were subjected to the in vitro assay described in Example 5 and the results are shown in FIG. 1.

Example 4

(77) Composition D was prepared using the same procedure described in Example 1 above except that RNase A-BNC shown in Scheme 5 was used instead of RNase A-Aco.

(78) RNase A-BNC was obtained following the same procedure described also in Example 1 except that boronic acid ester

(79) ##STR00025##
was used instead of cis-aconitic anhydride. See Scheme 5 above.

(80) Comparative Composition D was prepared following the same procedure used to prepare Composition D, except that RNase A-NC was used, which was prepared following the same procedure as RNase A-BNC except that

(81) ##STR00026##
was used instead of the boronic acid ester.

(82) Comparative Composition D was prepared following the same procedure used to prepare Composition D, except that no lipid-like compound was used.

(83) The three compositions were subjected to the in vitro assay described in Example 5.

Example 5

(84) Compositions A-D and Comparative Compositions A-D and D were tested for delivery of RNase A into B16F10 cancer cells.

(85) Cell Culture

(86) B16F10 cancer cells were cultured in Dulbecco's Modified Eagle Medium supplemented with 10% Fetal Bovine Serum and 1% penicillin/streptomycin at 37 C. in the presence of 5% CO.sub.2. For the protein transfection assay described below, cells were seeded in 96-well plates at a density of 10,000 cells per well a day prior to transfection.

(87) In Vitro Protein Transfection

(88) To evaluate protein delivery efficiency, Compositions A-D and Comparative Compositions A-D and D described in Examples 1-4 were added to B16F10 cancer cells and incubated at 37 C. for 24 hours. The protein concentration was 3.3 g/mL in PBS. Compound EC16-63, dissolved in the same volume of PBS, was used as a control. The cell viability was determined by the Alamar Blue assay after 24 hours of incubation. All transfection studies were performed in quadruplicate.

(89) For cells treated with Composition A, their viability was 45%; and for cells treated with Comparative Composition A, their viability was 100%. Note that low viability is an indication of high delivery efficiency. Unexpectedly, Composition A demonstrated high delivery efficiency, while Comparative Composition A was not delivered.

(90) For cells treated with Composition B, their viability was 50%; for cells treated with Comparative Composition B, their viability was 95%; for cells treated with Composition C, their viability was 20%; for cells treated with Comparative Composition C, their viability was 85%; and for cells treated with Compound EC16-63, their viability was 90%. Unexpectedly, Compositions B and C demonstrated much higher delivery efficiency than their corresponding Comparative Compositions B and C. As expected, Compound EC16-63 showed low cytotoxicity.

(91) For cells treated with Composition D (dosage at 4 g/mL), their viability was 30%; for cells treated with Comparative Composition D (dosage at 4 g/mL), their viability was 70%; for cells treated with Comparative Composition D (dosage at 4 g/mL), their viability was 95%. Unexpectedly, Composition D showed much higher delivery efficiency than their corresponding Comparative Compositions D and D.

OTHER EMBODIMENTS

(92) All of the features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

(93) From the above description, one skilled in the art can easily ascertain the essential characteristics of the present invention, and without departing from the spirit and scope thereof, can make various changes and modifications of the invention to adapt it to various usages and conditions. Thus, other embodiments are also within the scope of the following claims.