Patent Grant
9108931
The present invention relates to compounds, compositions, methods, and applications of a novel approach to administer bioactive compounds to patients in need thereof.
Radiotherapy and chemotherapy are well-established treatment methods for malignant diseases. Cells, which grow and divide rapidly, are most vulnerable to the effects of radiation and cytotoxic agents. Among those effected are tumor cells, and normal cells including hair and intestinal cells, and cells of the hemopoietic and immune systems. Damage to normal cells of the hemopoietic and immune systems by radiation and cytotoxic agents often has life-threatening consequences, and it limits the ability to administer a full therapeutic dose.
There has been extensive research to identify agents which will protect normal hemopoietic and immunologic cells from the effects of radiotherapy and chemotherapy, or aid in the reconstitution of cells suppressed by these therapies. For example, transforming growth factor beta-1 has been reported to be useful for protecting hematopoietic stem cells from the myelotoxicity of chemotherapeutic drugs or radiation therapy (U.S. Pat. No. 5,278,145 to Keller et al.). A lyophilized composition containing human albumin in thymosin alpha 1 was also reported to exert a preventative activity against progression of leukemia in mice whose immune systems were severely damaged by treatment with cytostatic agents or radiation treatment. Hemopoietic growth factors such as interleukin-3 and CSF have been used to potentiate immune response or assist in reconstituting normal blood following radiation-or-chemotherapy-induced hematopoietic cell suppression (WO 88/05469 to Anderson et al., U.S. Pat. No. 4,959,455 to Ciarletta et al; U.S. Pat. No. 4,999,291 to Souza).
Semina et al. (Radiatsionnaya Biologiya Radioekologiya 33(3), 1993; WO 89/06134) have shown that the levorotary (L) enantiomer of the dipeptide H-Glu-Trp-OH acts as an immunostimulant and can induce the proliferation of cells. As such, these dipeptides are useful in reconstituting hemopoietic and immune cells after chemotherapy or irradiation therapy.
Several peptides with immunoregulatory properties have been synthesized (for example, SU 1,582,393; EP 230,052; U.S. Pat. Nos. 4,190,646; 5,008,246; and 5,013,723). Many scientific laboratories have tried to develop methods for preparing synthetic derivatives of natural peptides, which are more active than their natural analogs (for example, EP 136,720; and EP 137,904).
Australian Patent No. AU-B-29308/89 (corresponding to WO 089/06134) teaches the preparation of Glu-Trp and its use for treating immune deficiency conditions. WO 93/08815 issued to Khavinson et al. discloses the peptide Glu-Trp and cyclic monomers and polymers thereof, for use in the treatment of immunosuppression.
Deigin et al., U.S. Pat. No. 6,051,683, issued Apr. 18, 2000 and U.S. Pat. No. 6,159,940 issued Dec. 12, 2002, describe immunoregulatory peptides of the general formula:
X-Glu-Trp-Y
that can stimulate the immune response and modulate hemopoiesis;
Deign et al., U.S. Pat. No. 5,736,519 issued Apr. 7, 1998; U.S. Pat. No. 6,103,699 issued Aug. 15, 2000 and U.S. Pat. No. 6,410,515 issued Jun. 25, 2003 describe immunoregulatory peptides of the general formula:
X-A-D-Trp-Y
for reconstitution of cells after radiation or chemotherapy, for inhibiting cell proliferation, and for immunosuppression, wherein X is hydrogen, glycine, alanine, leucine, isoleucine, valine, N-valine, proline, tyrosine, phenylalanine, tryptophan, D-alanine, D-leucine, D-isoleucine, D-valine, D-N-valine, D-proline, D-tyrosine, D-phenylalanine, D-tryptophan, γ-aminobutyric acid or ξ-aminocaproic acid; A is D-glutamic acid or D-γ-glutamic acid; and Y is glycine, alanine, leucine, isoleucine, valine, N-valine, proline, tyrosine, phenylalanine, tryptophan, D-alanine, D-leucine, D-isoleucine, D-valine, D-N-valine, D-proline, D-tyrosine, D-phenylalanine, D-tryptophn, γ-aminobutyric acid, ξ-aminocaproic acid, hydroxyl, or an amide group; and more particularly H-γ-DGlu-Trp-OH.
Deigin et al., U.S. Pat. No. 6,184,208, issued Feb. 6, 2001 and U.S. Pat. No. 6,248,716, issued Jun. 19, 2001 describe immunoregulatory peptides of the general formula:
X-Tyr-Y-Phe-Z-A
that reduce stress, stimulate weight gain, epithelium growth zone, wound healing and reparative and anabolic processes; wherein X is hydrogen, arginine, D-arginine, ornithine, D-ornithine, lysine, D-lysine, homoarginine, D-homoarginine, citrulline, D-citrulline; Tyr is tyrosine; Y is D-alanine, D-valine, D-leucine, D-isoleucine, D-phenylalanine, D-asparagine, D-tryptophan, D-proline, D-serine, D-threonine, D-tyrosine, D-hydroxyproline, D-cysteine, D-cysteyl-cysteine, D-methionine, D-lysine, D-homoarginine, D-arginine, D-histidine, D-aspartic acid, D-glutamic acid, D-β-alanine, or D-ornithine; Phe is phenylalanine; Z is alanine, D-alanine, valine, D-valine, leucine, D-leucine, isoleucine, D-isoleucine, phenylalanine, D-phenylalanine, asparagine, D-asparagine, glycine, glutamine, D-glutamine, tryptophan, D-tryptophan, proline, D-proline, serine, D-serine, threonine, D-threonine, tyrosine, D-tyrosine, hydroxyproline, D-hydroxyproline, cysteine, D-cysteine, cysteyl-cysteine, cysteine-D-cysteine, D-cysteyl cysteine, D-cysteine-D-cysteine, methionine, D-methionine, lysine, D-lysine, arginine, D-arginine, histidine, D-histidine, aspartic acid, D-aspartic acid, glutamic acid, D-glutamic acid, β-alanine, D-β-alanine, ornithine, or D-ornithine; and, A is hydroxyl or substituted amide (C1-C3); and more particularly H-Arg-Tyr-D-Ala-Phe-Gly-OH.
Compositions for the delivery, in particular, oral delivery, of active agents comprising a diketopiperazine-based system are described in several patents and patent applications owned by Emisphere Technologies, Inc., including for example, U.S. Pat. Nos. 6,663,898, 6,395,774, 6,331,318, 5,976,569 and 5,693,338, as well as U.S. Patent Application Nos. 20030198658, 20030155993 and 20030028250. In these compositions, the diketopiperazine is typically added as a separate component.
There remains a need for effective methods for delivering bioactive compounds to the body that may be readily tailored for any applications or for multiple applications.
The present invention represents a new platform for the therapeutic delivery of multifunctional bioactive compounds. The invention relates to molecules that possess an immunoregulatory portion linked to a stabilizer moiety. The stabilizer moiety acts as an effective carrier for the immunoregulator into the body. Further, the immunoregulator and stabilizer may be optionally linked to another functionally bioactive molecule. The bioactive molecule possesses either further immunoregulatory activity, activity which complements or synergizes with the immunoregulatory portion or a different therapeutic activity.
Accordingly, the present invention includes a multifunctional bioactive compound selected from one or more of a compound of Formula I:
wherein
The present invention further relates to pharmaceutical compositions comprising a multifunctional bioactive compound of the invention and a pharmaceutically acceptable carrier.
Also included in the present invention are methods of treating immune disorders, and optionally other disorders in the same subject, comprising administering an effective amount of a multifunctional bioactive compound of the invention to a subject in need thereof. Further, there is provided a use of a multifunctional bioactive compound of the invention to treat immune disorders, and optionally other disorders in the same subject, as well as a use of a multifunctional bioactive compound of the invention to prepare a medicament to treat immune disorders, and optionally other disorders in the same subject.
Other features and advantages of the present invention 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 invention are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.
The invention will now be described in relation to the drawings in which:
(i) Definitions
The following standard abbreviations for the amino acid residues are used throughout the specification: Ala—alanine; Arg—arginine; Asn—Asparagine; Asp—aspartic acid; Cys—cysteine; Glu—glutamic acid; iGlu—iso-glutamic acid; Gln—glutamine; His—histidine; Lys—lysine; Met—methionine; Ser—serine; Thr—threonine; Phe—phenylalanine; Gly—glycine; Ile—isoleucine; Leu—leucine; Pro—proline; Val—valine; Nval—N-valine; Trp—tryptophan; and Tyr—tyrosine.
The term “Ph” means phenyl.
The term “Bn” means benzyl.
The term “Me” means methyl.
The term “alkyl” as used herein means straight and/or branched chain alkyl groups containing from one to six carbon atoms and includes methyl, ethyl, propyl, isopropyl, t-butyl, pentyl, hexyl and the like.
The term “alkoxy” as used herein means straight and/or branched chain alkoxy groups containing from one to six carbon atoms and includes methoxy, ethoxy, propyloxyl, isopropyloxy, t-butoxy, hexyloxy and the like.
The term “alkenyl” as used herein means straight and/or branched chain alkenyl groups containing from two to six carbon atoms and one to three double bonds and includes vinyl, allyl, 1-butenyl, 2-hexenyl and the like.
The term “alkenyloxy” as used herein means straight and/or branched chain alkenyloxy groups containing from two to six carbon atoms and one to three double bonds and includes vinyloxy, allyloxy, propenyloxyl, butenyloxy, hexenyloxy and the like.
The term “alkylene” as used herein means bifunctional straight and/or branched alkyl radicals containing the specified number of carbon atoms.
The term “halo” as used herein means halogen and includes chloro, fluoro, bromo, iodo and the like.
The term “pharmaceutically acceptable” means compatible with the treatment of animals, in particular, humans.
The term “pharmaceutically acceptable salt” means an acid addition salt or basic addition salt which is suitable for or compatible with the treatment of animals, in particular, humans.
The term “pharmaceutically acceptable acid addition salt” as used herein means any non-toxic organic or inorganic salt of any base compound of the invention, or any of its intermediates. Basic compounds of the invention that may form an acid addition salt include those having a basic nitrogen, for example NH2 and NHC1-4alkyl. Illustrative inorganic acids which form suitable salts include hydrochloric, hydrobromic, sulfuric and phosphoric acids, as well as metal salts such as sodium monohydrogen orthophosphate and potassium hydrogen sulfate. Illustrative organic acids that form suitable salts include mono-, di-, and tricarboxylic acids such as glycolic, lactic, pyruvic, malonic, succinic, glutaric, fumaric, malic, tartaric, citric, ascorbic, maleic, benzoic, phenylacetic, cinnamic and salicylic acids, as well as sulfonic acids such as p-toluene sulfonic and methanesulfonic acids. Either the mono or di-acid salts can be formed, and such salts may exist in either a hydrated, solvated or substantially anhydrous form. In general, the acid addition salts of the compounds of the invention are more soluble in water and various hydrophilic organic solvents, and generally demonstrate higher melting points in comparison to their free base forms. The selection of the appropriate salt will be known to one skilled in the art. Other non-pharmaceutically acceptable salts, e.g. oxalates, may be used, for example, in the isolation of the compounds of the invention, for laboratory use, or for subsequent conversion to a pharmaceutically acceptable acid addition salt.
The term “pharmaceutically acceptable basic addition salt” as used herein means any non-toxic organic or inorganic base addition salt of any acid compounds of the invention. Illustrative inorganic bases which form suitable salts include lithium, sodium, potassium, calcium, magnesium or barium hydroxide. Illustrative organic bases which form suitable salts include aliphatic, alicyclic or aromatic organic amines such as methylamine, trimethylamine and picoline or ammonia. The selection of the appropriate salt will be known to a person skilled in the art.
The term “solvate” as used herein means a compound of the invention, or a pharmaceutically acceptable salt of a compound of the invention, wherein molecules of a suitable solvent are incorporated in the crystal lattice. A suitable solvent is physiologically tolerable at the dosage administered. Examples of suitable solvents are ethanol, water and the like. When water is the solvent, the molecule is referred to as a “hydrate”.
The term “compound(s) of the invention” as used herein means compound(s) of Formula I, and/or pharmaceutically acceptable salts, solvates and/or prodrugs thereof.
It is to be clear that the present invention includes pharmaceutically acceptable salts, solvates and prodrugs of compounds of the invention and mixtures comprising two or more compounds of the invention, pharmaceutically acceptable salts of the compounds of the invention (where applicable), pharmaceutically acceptable solvates of the compounds of the invention and prodrugs of the compounds of the invention.
The term an “effective amount” or a “sufficient amount” of an agent as used herein is that amount sufficient to effect beneficial or desired results, including clinical results, and, as such, an “effective amount” depends upon the context in which it is being applied. For example, in the context of administering an agent that acts as an immunomodulator, an effective amount of an agent is, for example, an amount sufficient to achieve such a modulation in immune response as compared to the response obtained without administration of the agent.
As used herein, and as well understood in the art, “treatment” is 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.
“Palliating” a disease or disorder means that the extent and/or undesirable clinical manifestations of a disorder or a disease state are lessened and/or time course of the progression is slowed or lengthened, as compared to not treating the disorder.
The term “modulate” as used herein includes the inhibition or suppression of a function or activity (such as immune response) as well as the enhancement of a function or activity.
To “inhibit” or “suppress” or “reduce” a function or activity, such as immune response, is to reduce the function or activity when compared to otherwise same 15 conditions except for a condition or parameter of interest, or alternatively, as compared to another conditions.
To “enhance” or “increase” or “stimulate” a function or activity, such as immune response, is to increase the function or activity when compared to otherwise same conditions except for a condition or parameter of interest, or alternatively, as compared to another conditions.
The term “subject” as used herein includes all members of the animal kingdom including human. The subject is preferably a human.
(ii) Compounds of the Invention
The present invention relates to compounds comprising an immunoregulatory portion, a stabilizer portion and, optionally a further functionally active portion. The portions are together via various linker groups to provide a multifunctional compound that can be used to treat multiple disorders in the same subject.
Accordingly, the present invention includes a multifunctional bioctive compound selected from one or more of a compound of Formula I:
wherein
In the compounds of the present invention, A is an immunoregulatory radical. The term “immunoregulatory radical” refers to any aromatic- or heteroaromatic-containing group having immuno- or hemosuppressive activity or immuno- or hemostimulative activity. Specifically, A is selected from the group consisting of Trp, Tyr, Phe, His, aryl and heteroaryl, where the aryl and heteroaryl groups may be optionally substituted with 1 to 6 substituents independently selected from the group consisting of halo, OH, OC1-6alkoxy, C1-6alkyl, C2-6alkenyl, C2-6alkenyloxy, NH2, NH(C1-6alkyl), N(C1-6alkyl)(C1-6alkyl), CN, CF3, OCF3, NO2, C(O)C1-6alkyl, C(O)OC1-6alkyl, SO2C1-6alkyl, SO2NH2, SO2NHC1-6alkyl, phenyl and C1-6alkylenephenyl, and where heteroaryl is an aromatic carbocyclic ring containing from 5 to 10 carbon atoms, in which 1 to 4 carbon atoms have been replaced with a heteroatom selected from one or more of O, S and N—R1, where R1 is selected from the group consisting of H, C1-6alkyl, C1-6alkylenearyl, C(O)C1-6alkyl, C(O)aryl, SO2C1-6alkyl and SO2aryl when the N atom is sp3 hybridized, or is a lone pair of electrons when the N atom is sp2 hybridized. In embodiments of the invention, A is selected from the group consisting of Trp, Tyr, Phe, His, aryl and heteroaryl, where the aryl and heteroaryl groups may be optionally substituted with 1 to 3 substituents independently selected from the group consisting of halo, OH, OC1-4alkoxy, C1-4alkyl, C2-4alkenyl, C2-4alkenyloxy, NH2, NH(C1-4alkyl), N(C1-4alkyl)(C1-4alkyl), CN, CF3, OCF3, NO2, C(O)C1-4alkyl, C(O)OC1-4alkyl, SO2C1-4alkyl, SO2NH2, SO2NHC1-4alkyl, phenyl and C1-4alkylenephenyl, and where heteroaryl is an aromatic carbocyclic ring containing from 5 to 10 carbon atoms, in which 1 to 3 carbon atoms have been replaced with a heteroatom selected from one or more of O, S and N—R1, where R1 is selected from the group consisting of H, C1-4alkyl, C1-2alkylenearyl, C(O)C1-4alkyl, C(O)aryl, SO2C1-4alkyl and SO2aryl when the N atom is sp3 hybridized, or is a lone pair of electrons when the N atom is sp2 hybridized. In further embodiments of the invention, A is selected from the group consisting of Trp, aryl and heteroaryl, wherein aryl is phenyl or naphthyl and heteroaryl is pyridinyl, imidazolyl, thienyl, furanyl, indolyl, isoquinolinyl, quinolinyl, benzothienyl, benzofuranyl, benzothiazolyl, thiazolo, benzooxazolyl, benzoisoxazolyl, benzoisothiazolyl or the like, with the indole ring of Trp, aryl and heteroaryl being unsubstituted or substituted with 1 to 2 substituents independently selected from the group consisting of halo, OH, OC1-4alkoxy, C1-4alkyl, C2-4alkenyl, C2-4alkenyloxy, NH2, NH(C1-4alkyl), N(C1-4alkyl)(C1-4alkyl), CN, CF3, OCF3, NO2, C(O)C1-4alkyl, C(O)OC1-4alkyl, SO2C1-4alkyl, SO2NH2, SO2NHC1-4alkyl, phenyl and C1-4alkylenephenyl.
When A is selected from Trp, Tyr, Phe or His, this group may be connected to the linker via the amine or the carboxyl group. When not connected to the linker, the free amine or carboxyl group may be derivativized. For example, the amine may be mono or dialkylated with C1-6alkyl, acylated with a C(O)C1-6alkyl or converted to NH3+ by addition of a pharmaceutically acceptable acid. Further, the carboxyl group may be esterified, for example as a C1-6alkyl ester, converted to the corresponding amide which may also be mono- or diesterified with C1-6alkyl, converted to its corresponding hydrazine or to its corresponding basic addition salt. In another aspect of the present invention, it has been found that when A is Trp, the immuno- and hemomodulating activity of this group may be controlled by the stereochemistry at the α-carbon. For example, when the stereochemistry is in the D-configuration, this group may possess immuno- and hemodepressant activity and when it is in the L-configuration it may possess immuno- and hemostimulant activity. Accordingly, the immunomodulatory activity of the molecules of the present invention can be controlled and tailored for specific uses and combination therapies.
In an embodiment of the invention, A is a group having the formula II:
wherein R3 is selected from the group consisting of H, OC1-6alkyl, NH2, NHC1-6alkyl, N(C1-6alkyl)2, NHNH2 and OY, where Y is a pharmaceutically acceptable cation; R4 is 1 to 4 substituents which are independently selected from the group consisting of H, halo, OH, OC1-6alkoxy, C1-6alkyl, C1-6alkenyl, C1-6alkenyloxy, NH2, NH(C1-6alkyl), N(C1-6alkyl)(C1-6alkyl), CN, CF3, OCF3, NO2, C(O)C1-6alkyl, C(O)OC1-6alkyl, SO2C1-6alkyl, SO2NH2, SO2NHC1-6alkyl, phenyl and C1-6alkylenephenyl; and * is the L or D configuration or mixtures thereof.
The present invention includes compounds of Formula I wherein R3 is selected from the group consisting of H, OC1-6alkyl, NH2, NHC1-6alkyl, N(C1-6alkyl)2, NHNH2 and OY, where Y is a pharmaceutically acceptable cation. In embodiments of the invention, R3 is selected from the group consisting of H, OC1-4alkyl, NH2, NHC1-4alkyl, N(C1-4alkyl)2, NHNH2 and OY. In further embodiments of the invention, R3 is selected from the group consisting of H, Me, NH2, NHMe, NMe2, NHNH2 and OY. The cation “Y” may be any pharmaceutically acceptable cation, for example Na+, K+ and Zn2+.
The compounds of Formula I also include those in which R4 is 1 to 4 substituents which are independently selected from the group consisting of H, halo, OH, OC1-6alkoxy, C1-6alkyl, C1-6alkenyl, C1-6alkenyloxy, NH2, NH(C1-6alkyl), N(C1-6alkyl)(C1-6alkyl), CN, CF3, OCF3, NO2, C(O)C1-6alkyl, C(O)OC1-6alkyl, SO2C1-6alkyl, SO2NH2, SO2NHC1-6alkyl, phenyl and C1-6alkylenephenyl. In embodiments of the invention, R4 is 1 to 3 substituents which are independently selected from the group consisting of H, halo, OH, OC1-4alkoxy, C1-4alkyl, C2-4alkenyl, C2-4alkenyloxy, NH2, NH(C1-4alkyl), N(C1-4alkyl)(C1-4alkyl), CN, CF3, OCF3, NO2, C(O)C1-4alkyl, C(O)OC1-4alkyl, SO2C1-4alkyl, SO2NH2, SO2NHC1-4alkyl, phenyl and C1-4alkylenephenyl. In further embodiments of the invention, R4 is 1 to 3 substituents which are independently selected from the group consisting of H, halo, OH, OMe, Me, vinyl, vinyloxy, NH2, NHMe, NHMe2, CN, CF3, OCF3, NO2, C(O)Me, C(O)OMe, SO2Me, SO2NH2, SO2NHMe, phenyl and benzyl. In still further embodiments of the invention, R4 is a substituent selected from the group consisting of H, halo, OH, OMe, Me, vinyl, vinyloxy, NH2, NHMe, NHMe2, CN, CF3, OCF3, NO2, C(O)Me, C(O)OMe, SO2Me, SO2NH2, SO2NHMe, phenyl and benzyl. In even further embodiments of the invention, R4 is H.
In the multifunctional bioactive compounds of the present invention, each of L1 and L2 is a linker group independently selected from the group consisting of a single bond, —C(O)—, —C(O)NR2—, —NR2C(O)—, —NR2—, —C(O)—O—, —OC(O)—, —S—S—, SO2NR2—, NR2SO2, —S— and —O—, where R2 is selected from the group consisting of H, C1-6alkyl, C1-6alkylenearyl, C(O)C1-6alkyl, C(O)aryl, SO2C1-6alkyl and SO2aryl. In embodiments of the invention, each of L1 and L2 is independently selected from the group consisting of a single bond, —C(O)—, —C(O)NR2—, —NR2C(O)—, —NR2—, —C(O)—O—, —OC(O)— and —O—. In further embodiments of the invention, each of L1 and L2 is independently selected from the group consisting of —C(O)—, —NR2—, —C(O)NR2— and —NR2C(O)—. In other embodiments of the invention, R2 is selected from the group consisting of H, C1-4alkyl, C1-4alkylenearyl, C(O)C1-4alkyl, C(O)Ph, SO2C1-4alkyl and SO2Ph. In still further embodiments of the invention, R2 is selected from the group consisting of H, Me, Bn, C(O)Me, C(O)Ph, SO2Me and SO2Ph. In even further embodiments of the invention, R2 is H.
In the bioactive compounds of the invention, m is an integer between 1 and 50. In embodiments of the invention, m is an integer between 1 and 25. In further embodiments of the invention, m is an integer between 1 and 10. In still further embodiments of the invention, m is an integer between 1 and 6.
The multifunctional bioactive compounds of the invention include those where n is an integer between 0 and 50. In embodiments of the invention, n is an integer between 0 and 25. In further embodiments of the invention, n is an integer between 0 and 10.
The compounds of Formula I, include those in which R5 is selected from the group consisting of H and C1-6alkyl and * represents the D or L configuration or mixtures thereof. In embodiments of the invention, R5 is selected from H and C1-4alkyl, specifically H and Me. In further embodiments of the invention, both * are substantially in the D configuration or both are substantially in the L configuration.
The multifunctional bioactive compounds of the invention also include those where D is selected from the group consisting of H, C1-6alkyl, any side chain of an amino acid and any functionally bioactive molecule. In embodiments of the invention, D is selected from the group consisting of H, C1-4alkyl, any side chain of an amino acid and any functionally active molecule. By functionally bioactive molecule, it is meant any molecule having a pharmacological effect in the subject. This pharmacological effect may be one which complements, enhances or synergizes with the immunoregulatory group A, or it may provide another therapeutic action so that when the bioactive compounds of the invention are administered to the subject, combination therapy is effected. More than one functionally bioactive molecule may be used. Examples of functionally active molecules include, but are not limited to, adjuvants such as palmitoyl; analgesics such as peptide analgesics; opiates and antidotes such as dermorphin, morphine, naloxone and derivatives thereof; synthetic vaccines such as antigenic determinants—T- and B-epitopes; antibiotics, such as fusidic acid, pharmaceutical pharmacophores including small molecules such as methotrexate, diclophenac, ibuprophen, indometacine, naproxen, ketoprofen; sugars; lipids; and nucleotides.
In an embodiment of the present invention, the multifunctional bioactive compounds of Formula I have the following formula:
wherein L1, L2, D, R3, R4, R5, m, n and * have the meanings provided above.
In a further embodiment of the invention, the multifunctional bioactive compounds of Formula I have the following formula:
wherein L2, D, R3, R4, R5, m, n and * have the meanings provided above.
Examples of multifunctional bioactive compounds and pharmaceutically acceptable salts, solvates and prodrugs thereof, representing specific embodiments of the present invention are shown in Tables 1 and 9.
All of the compounds of the invention have more than one asymmetric centre. Where the compounds according to the invention possess more than one asymmetric centre, they may exist as diastereomers. It is to be understood that all such isomers and mixtures thereof in any proportion are encompassed within the scope of the present invention. It is to be understood that while the relative stereochemistry of the compounds of the invention may be as shown in any given compound shown herein, such compounds of the invention may also contain certain amounts (e.g. less than 20%, preferably less than 10%, more preferably less than 5%) of compounds of the invention having alternate stereochemistry.
The compounds of the invention can be prepared from known starting materials using procedures known in the art. Generally, the compounds are prepared by the coupling of two or more entities together, for example, using standard coupling chemistry (e.g. formation of peptide bonds, amide linkages, disulfide linkages, ester linkages, etc.). The diketopiperazine moiety may be prepared using known chemistry. For example, diketopiperazines can be formed by cyclodimerization of amino acid ester derivatives as described by Katchalski et al. in J. Amer. Chem. Soc., 68, 879-880 (1946), by cyclization of dipeptide ester derivatives, or by thermal dehydration of amino acid derivatives and high boiling solvents as described by Kopple et al. in J. Org. Chem., 33 (2), 862-864 (1968).
In some cases, the chemistries used to prepare the compounds of the invention may have to be modified, for instance, by use of protective groups, to prevent side reactions due to reactive groups, such as reactive groups attached as substituents. This may be achieved by means of conventional protecting groups, for example as described in “Protective Groups in Organic Chemistry” McOmie, J. F. W. Ed., Plenum Press, 1973 and in Greene, T. W. and Wuts, P. G. M., “Protective Groups in Organic Synthesis”, John Wiley & Sons, 3rd Edition, 1999.
The formation of a desired compound salt is achieved using standard techniques. For example, the neutral compound is treated with an acid or base in a suitable solvent and the formed salt is isolated by filtration, extraction or any other suitable method.
The formation of solvates of the compounds of the invention will vary depending on the compound and the solvate. In general, solvates are formed by dissolving the compound in the appropriate solvent and isolating the solvate by cooling or using an antisolvent. The solvate is typically dried or azeotroped under ambient conditions.
The present invention includes within its scope, prodrugs of the compounds of the invention. In general, such prodrugs will be functional derivatives of a compound of the invention which are readily convertible in vivo into the compound from which it is notionally derived. Prodrugs of the compounds of the invention may be conventional esters formed with available hydroxy, thiol, amino or carboxyl group. For example, an available OH or NH2 group in a compound of the invention may be acylated using an activated acid in the presence of a base, and optionally, in inert solvent (e.g. an acid chloride in pyridine). Some common esters which have been utilized as prodrugs are phenyl esters, aliphatic (C8-C24) esters, acyloxymethyl esters, carbamates and amino acid esters. In further embodiments, the prodrugs of the compounds of the invention are those in which one or more of the hydroxy groups in the compounds is masked as groups which can be converted to hydroxy groups in vivo. Conventional procedures for the selection and preparation of suitable prodrugs are described, for example, in “Design of Prodrugs” ed. H. Bundgaard, Elsevier, 1985.
The present invention includes radiolabeled forms of compounds of the invention, for example, compounds of the invention labeled by incorporation within the structure 3H or 14C or a radioactive halogen such as 125I. A radiolabeled compound of the invention may be prepared using standard methods known in the art. For example, tritium may be incorporated into a compound of the invention using standard techniques, for example, by hydrogenation of a suitable precursor to a compound of the invention using tritium gas and a catalyst. Alternatively, a compound of the invention containing radioactive iodo may be prepared from the corresponding trialkyltin (suitably trimethyltin) derivative using standard iodination conditions, such as [125I] sodium iodide in the presence of chloramine-T in a suitable solvent, such as dimethylformamide. The trialkyltin compound may be prepared from the corresponding non-radioactive halo, suitably iodo, compound using standard palladium-catalyzed stannylation conditions, for example hexamethylditin in the presence of tetrakis(triphenylphosphine) palladium (0) in an inert solvent, such as dioxane, and at elevated temperatures, suitably about 50 to 100° C.
(iii) Uses
The present invention provides novel compounds of the Formula I. Accordingly, the present invention includes all uses of the compounds of the invention including their use in therapeutic methods and pharmaceutical compositions, their use in diagnostic assays and their use as research tools.
The present invention in particular relates to pharmaceutical compositions comprising a multifunctional bioactive compound of the invention and a pharmaceutically acceptable carrier.
Also included in the present invention are methods of treating immune disorders, and optionally other disorders in the same subject, comprising administering an effective amount of a multifunctional bioactive compound of the invention to a subject in need thereof. Further, there is provided a use of a multifunctional bioactive compound of the invention to treat immune disorders, and optionally other disorders in the same subject, as well as a use of a multifunctional bioactive compound of the invention to prepare a medicament to treat immune disorders, and optionally other disorders in the same subject.
In one aspect, the invention provides a method of modulating the immune system and/or hemopoiesis in an animal comprising administering an effective amount of a multifunctional bioactive compound of the invention to a subject in need thereof.
In an embodiment of the invention, there is provided a method of stimulating the immune system comprising administering an effective amount of a multifunctional bioactive compound of the invention to a subject in need thereof. In another embodiment, the invention provides a method of restoring hemopoiesis in an animal with impaired hemopoiesis, for example caused by irradiation or cytostatic agents, comprising administering an effective amount of a multifunctional bioactive compound of the invention to a subject in need thereof.
In yet another embodiment, the invention provides a method of treating hemopoietic disorders, for example, without limiting to, immune cytopenia, multiple myeloma, chronic lymphoid leucosis, lymphocytic lymphomas, lymphosarcomas and in particular, B-cellular lymphoid leucosis, comprising administering an effective amount of a multifunctional bioctive compound of the invention to a subject in need thereof.
In another embodiment, the invention provides a method for treating immune and/or hemopoietic disorders such as cancer in an animal comprising administering an effective amount of a multifunctional bioactive compound of the invention to a subject in need thereof, possibly in combination with a cytostatic agent. The cytostatic agent may be, for example, oxyurea or hyperthermia.
In yet another embodiment, the invention also relates to a method of immunosuppressing an immune system in an animal comprising administering an effective amount of a multifunctional bioactive compound of the invention to a subject in need thereof. In one embodiment, the compound may be administered prior to an organ or bone marrow transplant. The immunoregulatory properties of the compounds of the invention may be controlled, for example, by the stereochemistry in the “A” portion and “*” of the compound.
The compounds of the invention may be used in the form of the free base, free acid, in the form of salts, solvates and/or prodrugs. All forms are within the scope of the invention.
In accordance with the methods of the invention, the described compounds, salts, prodrugs or solvates thereof may be administered to a patient in a variety of forms depending on the selected route of administration, as will be understood by those skilled in the art. The compositions of the invention may be administered, for example, by oral, parenteral, buccal, sublingual, nasal, rectal, patch, pump or transdermal (topical) administration and the pharmaceutical compositions formulated accordingly. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, intrapulmonary, intrathecal, rectal and topical modes of administration. Parenteral administration may be administered by continuous infusion over a selected period of time.
A compound of the invention 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 compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the compound of the invention may be incorporated with excipient and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
A compound of the invention may also be administered parenterally. Solutions of a compound of the invention 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 (2003—20th edition) and in The United States Pharmacopeia: The National Formulary (USP 24 NF19) published in 1999.
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 forms must be sterile and must be fluid to the extent that easy syringability exists. Ampoules are convenient unit dosages.
Compositions for nasal administration 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 atomizing 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.
Compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, wherein the active ingredient is formulated with a carrier such as sugar, acacia, tragacanth, or gelatin and glycerine. Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base such as cocoa butter.
Compositions for topical administration may include, for example, propylene glycol, isopropyl alcohol, mineral oil and glycerin. Preparations suitable for topical administration include liquid or semi-liquid preparations such as liniments, lotions, applicants, oil-in-water or water-in-oil emulsions such as creams, ointments or pastes; or solutions or suspensions such as drops. In addition to the aforementioned ingredients, the topical preparations may include one or more additional ingredients such as diluents, buffers, flavoring agents, binders, surface active agents, thickeners, lubricants, preservatives, e.g. methyl hydroxybenzoate (including anti-oxidants), emulsifying agents and the like.
Sustained or direct release compositions can be formulated, e.g. liposomes or those wherein the active compound is protected with differentially degradable coatings, such as by microencapsulation, multiple coatings, etc. It is also possible to freeze-dry the compounds of the invention and use the lypolizates obtained, for example, for the preparation of products for injection.
The compounds of the invention may be administered to an subject alone or in combination with pharmaceutically acceptable carriers, as noted above, the proportion of which is determined by the solubility and chemical nature of the compound, chosen route of administration and standard pharmaceutical practice.
The dosage of the compounds and/or compositions of the invention 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 subject to be treated. One of skilled in the art can determine the appropriate dosage based on the above factors. For example, in the topical treatment, ointments, creams or lotions containing from 1-1000 μg/g of a compound of the invention may be administered. Oral preparations may be formulated, preferably as tablets, capsules, or drops, containing from 0.5-1000 μg of a compound of the invention per dosage unit. The compounds of the invention may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response. For ex vivo treatment of cells over a short period, for example for 30 minutes to 1 hour or longer, higher doses of compound may be used than for long term in vivo therapy.
In addition to the above-mentioned therapeutic uses, the compounds of the invention are also useful in diagnostic assays, screening assays and as research tools.
In diagnostic assays, the compounds of the invention may be useful in identifying or detecting an immune disorder. In such an embodiment, the compounds of the invention may be radiolabeled (as hereinbefore described) and contacted with a population of cells. The presence of the radiolabel on the cells may indicate an immune disorder.
In screening assays, the compounds of the invention may be used to identify other compounds that modulate immune responses. In such assays, the compounds may also be radiolabeled.
The following non-limiting examples are illustrative of the present invention:
Materials and Methods
NMR experiments were performed on Bruker Avance DRX 500 spectrometer. The spectra were acquired in 0.6 μl (CD3)2SO at 30° C. (99.95% Deuterium, Deiton, S.Peterburg). A relaxation delay of 5.0 s was used. The 1H chemical shifts were determined relative to those (arbitrary chosen as 2.5 ppm at 30° C.) of the (CH3)2SO signal.
HPLC analyses were performed on System Gold Beckman analytical gradient chromatographic device. Column Ultrasphere-ODS, 5μ, 205×4.6 mm. Detection—UV spectrophotometer, λ214 nm. Ambient temperature. Gradient 0.02M triethylammonium phosphate buffer (pH=3.0) in acetonitrile (from 0% buffer A to 100% buffer B). Buffer A—15% of 0.02M triethylammonium phosphate in acetonitrile. Buffer B—50% of 0.02M triethylammonium phosphate in acetonitrile.
Mass spectra were acquired on a VISION 2000 MALDI mass spectrometer.
In a like manner, the following additional compounds were prepared. Mass spectrum data for some of these compounds are provided below.
The effect of tested substances on intact bone marrow was studied in vivo. The peptides were introduced into mice in different manners: injected subcutaneously, intraperitoneally (IP) or introduced per os to intact donor mice, in the doses of 10-1000 μg/kg. Two days after administration of preparation, the mice were killed. Bone marrow suspension was prepared and injected intravenously to lethally irradiated mice. The colony forming activity was evaluated at day 8. Test animals were (CBA×C57 B1) F1 mice (30 mice per trial, average from 3 tests).
As can be seen from Table 2, it was found that introduction of thymodepressin and the novel cyclopeptides of the present invention into intact mice decreased the CFU-S population in the bone marrow.
A comparison of the activities of the novel cyclopeptides of the present invention and thymodepressin on suppression of the CFU-S population in the bone marrow is shown in Table 3.
A study to compare the activity of Neogen and the new cyclic peptides of the present invention in reducing the harmful effects of ionizing radiation was performed.
In this study, the method of exogenous spleen colonies was applied. A suspension of intact bone marrow cells was irradiated ex vivo in the dose of 1 Gy. Different doses of Neogen or cyclic peptides were injected IP, IM, subcutaneous or introduced per os to lethally irradiated recipients within an hour after the injection of irradiated bone marrow. Colonies were counted on day 8. All data in each group was the mean of three tests.
The data in Table 4 shows that Neogen can stimulate the regeneration process after the detrimental effect of radiation on hemopoietic precursor cells. This process was shown to be effective with IM or IP injection, but not via per os administration. New cyclic peptides under investigation possessed the same range of activity during systemic administration and are orally active at the dose range of 10-100 μg/kg.
Adjuvant activity was tested on 5 groups of mice (C57Bl6). Each group consisted of 5 animals. Three immunizations were performed:
First Immunization:
The same set 21 days after first immunization by injection of 12.5 micrograms of ovalbumin/mouse to groups 2-5.
Third Immunization:
The same set 35 days after first immunization by injection of 12.5 micrograms of ovalbumin/mouse to groups 2-5.
On day 42, the total blood from each mouse was collected. The blood was pooled from each group of mice. The stimulation index of each group was compared. The stimulation index was calculated as a ratio between optical density (OD=1) of diluted pools of groups 3-5 to the optical density (OD=1) of diluted pools of group 2 (control with no adjuvant).
As can be seen in Table 5, the results of this experiment show that cyclopeptide Cyclo-L-Ala-L-Glu-(L-Trp-ONa) alone, even without further modification by coupling sugar or palmitoyl function, possesses adjuvant activity and increases by 74% titers of antibodies to ovalbumin.
Adjuvant activity was tested on 6 groups of mice. Each group consisted of 5 animals. Three immunizations were performed as follows:
First Immunization:
The same set 21 days after first immunization by injection of 12.5 micrograms of ovalbumin/mouse to mouse nos. 6 to 30.
Third Immunization:
The same set 35 days after first immunization by injection of 12.5 microgram of ovalbumin/mouse to mouse nos. 6 to 30.
On day 42, blood samples from each mouse were collected. The stimulation index of each group was compared. The stimulation index was calculated by dividing the test pooled sera dilution yielding OD=1 by the dilution of pooled sera from mice immunized without the adjuvant, yielding OD=1. The results are shown in Table 6.
Adjuvant Activities of cyclo-L-Ala-L-Glu-(L-Trp-OH) (Compound 17), cyclo [L-Lys(Palmitoyil)-L-Glu(D-Trp-OH)] (Compound 26a), cyclo [D-Lys(Palmitoyil)-D-Glu(D-Trp-OH)] (Compound 26b), cyclo-L-Lys(N-acetyl-Glycosamine-N-acetyl-muramil)-L-Glu-(L-Trp-OH) (Compound 33a), and cyclo-D-Lys(N-acetyl-Glycosamine-N-acetyl-muramil)-D-Glu-(L-Trp-OH) (Compound 33b)
Stage 1:
Adjuvant activity was tested on 11 groups of Balb/c mice (obtained from Stolbovaya Breeding Station). Each group consisted of 7 animals. Three immunizations were performed as follows:
First Immunization:
The same set 14 days after first immunization by injection of 12.5 micrograms of ovalbumin/mouse to mouse nos. 1 to 77.
Third Immunization:
The same set 28 days after first immunization by injection of 12.5 micrograms of ovalbumin/mouse to mouse nos. 1 to 77.
On day 35 after first immunization, blood samples from each mouse were collected.
Enzyme-Linked Immunosorbent Assay (ELISA):
(i) Evaluation of Peripheral Opioid Activity
Peripheral opioid activity of the peptides was assessed on the basis of the ability to inhibit the electrically-induced contractions of isolated guinea pig ileum (GPI) (Kosterlitz, H. W. et al., “The effect of adrenaline, noradrenaline and isoprenaline on inhibition a- and b-adrenoreceptors in the longitudinal muscle of the guinea pig ileum”, Brit. J. Pharmacol., Vol. 39., Pages 398 to 413, 1970).
The segment of GPI about 1 cm long was placed into a 10-ml organ bath containing Krebs solution at 34° C. The composition of the Krebs solution was (mM): NaCl—118; KCl—4.70; CaCl2—2.52; KH2PO4—0.93; MgSO4—1.27; NaHCO3—25; glucose—11.0. Resting tension of the organ was 1 g. The segment of GPI was stimulated by single pulses duration 1 ms with 0.1 Hz at 80 V. The solution with isolated organs was constantly aerated. The contractions were recorded in an isometrical mode by a sensor K 30 (Hugo Sachs Elektronic KG) with paper register Rikadenki-series (Japan).
The substances tested were dissolved in distilled water and added cumulatively to the organ bath at a volume of 5 to 30 mcl. Each subsequent substance was added after the isolated organs were washed 3 or 4 times for 12-15 min. On the basis of the data obtained, dose-effects curves were plotted and the activity of substances was expressed by IC50 or pD2. The pD2 index was equal to a negative decimal logarithm of the substance concentration causing a 50% of the maximal effect.
Statistical treatment of the results was carried out by the Student's t-test.
New dermorphin analogues were tested in the standard model of Guinea pig ileum binding test. For each molecule, the EC50 was determined as concentration of substance causing the reduction of contraction amplitude by the 50% from basic level.
The standard preparation in all experiments was dermorphin. For confirmation of opioid activity, the specific antagonist naloxson was used in concentration of 10−5M. Each molecule was tested in 5 independent replications and relative activity was calculated as negative logarithm of EC50 (pD2).
As can be seen in Table 9, all tested peptides have different levels of opioid activities, in the range of 10−9 to 10−5 M, with the exception of H-Tyr-Tyr-Pro-Ser-NH2 (Compound 51) and D-Ala-D-Glu-(D-Trp)-OH (Compound 54).
(ii) Analgesic Activity of New Opioid Peptide Analogues.
Two hundred and twenty mice F1(CBA×C57D16) first generation hybrids were used for testing of analgesic activity using the “tail flick” test. The water temperature was at 48° C. The maximal effect was for the period of 30 seconds. All peptides were injected intraperitoneally in the doses of 5, 10 or 20 mg/kg. Analgesic effect was estimated in the time interval of 15 to 120 min after peptide injection. Student's t-criterion was used for statistical calculations of the results. Statistical significance were at the level of p≦0.05. The results of the tests are shown in Table 10.
(iii) Analgesic Activity of Dermorphin and Analogues After Oral Administration and Intraperitoneal Administration.
For estimation of oral activity of novel cyclopeptides of the present invention, the “tail flick” test was used. The results were compared with those obtained using intraperitoneal administration.
Antinociceptive activity of the peptides of the present invention was assessed in experiments on BALB/c male mice weighing 22 to 24 g. The peptides were dissolved in saline and administered intraperitoneally or intragastrally.
The “Tail flick” test (D'Amour, F. E. et al. “A Method for Determining Loss of Pain Sensation”, J. Pharmacol. Exp. Ther., Vol. 72, Pages 74 to 79, 1941) was performed on an analgesimeter type 812, Hugo Sachs Electronick KG. Antinociceptive activity was defined as the absence of tail flick response on stimulation with a focused bundle of heat radiation with 6 sec at baseline response of 2.0-3.0 seconds.
Statistical treatment of the results was carried out by the Student's paired t-test. The results of these testing are shown in Tables 11 and 12. As can be seen from the experiments, only cycloanalogues on neuropeptides are active in oral (intragastrally) administration.
While the present invention has been described with reference to what are presently considered to be the preferred examples, it is to be understood that the invention is not limited to the disclosed examples. To the contrary, the invention is intended to cover various modifications and equivalent arrangements included within the spirit and scope of the appended claims.
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. Where a term in the present application is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term.
15
16
17
18
19
20
21
4.8
4.5
4.5
4.8
4.8
4.2
4.8
29
30
31
32
33
34
35
4.8
4.7
5.1
4.7
4.8
4.7
4.8
36
37
38
39
40
41
5.1
5.4
4.5
3.8
3.5
5.1
5.4
22
23
24
25
26
27
28
5.1
4.8
4.4
4.8
5.1
4.8
4.8
5.4
42
43
44
45
46
47
48
5.7
5.5
5.5
5.8
5.5
5.7
5.7
5.5
49
50
51
21
53
54
55
4.4
4.8
5.1
4.5
4.4
5.1
5.1
5.1
8
9
10
11
12
13
14
6.2
6.2
5.9
6.2
6.2
6.2
6.2
5.9
1
2
3
4
5
6
7
3.9
4.1
4.2
4.2
3.9
4.5
3.9
3.5
51
H-Tyr-Tyr-Pro-Ser-NH
2
54
D-Ala-D-Glu-(D-Trp)-OH
11.0 ± 1.0
14.4 ± 3.0
17.2 ± 2.4
13.2 ± 3.2
11.7 ± 1.0
16.6 ± 2.2
17.0 ± 2.3
10.2 ± 1.2
9.0 ± 1.7
10.3 ± 2.6
14.3 ± 3.7
10.8 ± 1.9
6.4 ± 1.5
5.9 ± 0.7
10.7 ± 0.8
12.2 ± 2.6
7.2 ± 1.2
6.9 ± 1.4
6.8 ± 0.6
6.3 ± 0.7
8.4 ± 1.4
7.7 ± 1.7
6.7 ± 1.0
7.8 ± 0.5
8.3 ± 0.8
8.7 ± 1.2
9.5 ± 1.2
9.7 ± 1.3
8.2 ± 1.7
11.6 ± 1.7
15.5 ± 1.6
15.7 ± 1.2
13.0 ± 1.5
9.9 ± 1.5
6.8 ± 0.5
1.4 ± 1.6
18.1 ± 2.3
16.8 ± 2.2
12.7 ± 1.8
11.7 ± 1.2
6.3 ± 0.6
3.7* ± 0.5
3.9* ± 0.5
4.1* ± 0.5
4.0* ± 0.5
4.5* ± 0.1
4.1* ± 0.5
5.5* ± 0.5
5.5* ± 0.4
5.5* ± 0.5
5.6* ± 0.4
6.0* ± 0
5.6* ± 0.4
5.5* ± 0.6
5.2* ± 0.6
5.6* ± 0.4
4.8* ± 0.6
5.3* ± 0.7
5.2* ± 0.6
This application is a Divisional of U.S. patent application Ser. No. 12/376,138, filed Oct. 22, 2009, which is a National Phase Entry of PCT/CA2007/001357, filed Aug. 1, 2007, which claims priority to U.S. Provisional Patent Application No. 60/835,428 filed Aug. 4, 2006, the entire specification, claims and drawings of each of which are incorporated herewith by reference.
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| Number | Date | Country | |
|---|---|---|---|
| 20140179708 A1 | Jun 2014 | US |
| Number | Date | Country | |
|---|---|---|---|
| 60835428 | Aug 2006 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | 12376138 | US | |
| Child | 14134418 | US |
