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DISEASE DETECTION AND TREATMENT THROUGH ACTIVATION OF COMPOUNDS USING EXTERNAL ENERGY

20200147216 ยท 2020-05-14

    Inventors

    Cpc classification

    International classification

    Abstract

    Described herein are compounds for the detection, diagnosis, and treatment of specific diseased tissues, including hyper-proliferative tissues such as tumors, and other tissue diseased with microbial and/or infectious species, using energy-activation methods. In particular, compounds sensitive to externally applied energy, including light and/or ultrasound; that also specifically accumulate in diseased target tissue, are provided.

    Claims

    1-49. (canceled)

    50. A method for treating cancer in a subject using one or more of sonodynamic therapy and photodynamic therapy, the method comprising administering to the subject an agent comprising a mixture a first compound, a second compound, a third compound and a fourth compound, each compound having an independent structural formula IV: ##STR00006## wherein M is Sn(IV), X is Cl, m is 2, Y.sub.1 and Y.sub.2 are OH, and NR.sub.4R.sub.5 is a peptide amide; wherein the first, second, third and fourth compounds are present in the agent in a weight ratio of approximately 4:2:1:1, and subjecting the subject to one or more of ultrasound and red light.

    51. The method according to claim 50, wherein the first compound is Sn(IV) chlorin e6 gly-gly amide dihydroxide sodium salt.

    52. The method according to claim 50, wherein the second compound is Sn(IV) chlorin e6 gly-gly-gly-gly amide dihydroxide sodium salt.

    53. The method according to claim 50, wherein the third compound is Sn(IV) chlorin e6 Taurine amide dihydroxide sodium salt.

    54. The method according to claim 50, wherein the fourth compound is Sn(IV) chlorine e6 L-serine amide dihydroxide sodium salt.

    55. The method according to claim 50, wherein the fourth compound is Sn(IV) chlorine e6 lycine amide dihydroxide sodium salt.

    56. The method according to claim 50, wherein the first compound is Sn(IV) chlorin e6 gly-gly amide dihydroxide sodium salt, the second compound is Sn(IV) chlorin e6 gly-gly-gly-gly amide dihydroxide sodium salt, the third compound is Sn(IV) chlorin e6 Taurine amide dihydroxide sodium salt, and the fourth compound is Sn(IV) chlorine e6 L-serine amide dihydroxide sodium salt.

    57. The method according to claim 50, wherein the first compound is Sn(IV) chlorin e6 gly-gly amide dihydroxide sodium salt, the second compound is Sn(IV) chlorin e6 gly-gly-gly-gly amide dihydroxide sodium salt, the third compound is Sn(IV) chlorin e6 Taurine amide dihydroxide sodium salt, and the fourth compound is Sn(IV) chlorine e6 lycine amide dihydroxide sodium salt.

    58. A method for treating cancer in a subject using one or more of sonodynamic therapy and photodynamic therapy, the method comprising administering to the subject an agent comprising a mixture a first compound, a second compound, a third compound a fourth compound, and a fifth compound, each compound having an independent structural formula IV: ##STR00007## wherein M is Sn(IV), X is Cl, m is 2, Y.sub.1 and Y.sub.2 are OH, and NR.sub.4R.sub.5 is a peptide amide; wherein the first, second, third and fourth compounds are present in the agent in a weight ratio of approximately 4:2:1:1:1, and subjecting the subject to one or more of ultrasound and red light.

    59. The method according to claim 58, wherein the first compound is Sn(IV) chlorin e6 gly-gly amide dihydroxide sodium salt.

    60. The method according to claim 58, wherein the second compound is Sn(IV) chlorin e6 gly-gly-gly-gly amide dihydroxide sodium salt.

    61. The method according to claim 58, wherein the third compound is Sn(IV) chlorin e6 Taurine amide dihydroxide sodium salt.

    62. The method according to claim 58, wherein the fourth compound is Sn(IV) chlorine e6 L-serine amide dihydroxide sodium salt.

    63. The method according to claim 58, wherein the fifth compound is Sn(IV) chlorine e6 lycine amide dihydroxide sodium salt.

    64. The method according to claim 50, wherein the first compound is Sn(IV) chlorin e6 gly-gly amide dihydroxide sodium salt, the second compound is Sn(IV) chlorin e6 gly-gly-gly-gly amide dihydroxide sodium salt, the third compound is Sn(IV) chlorin e6 Taurine amide dihydroxide sodium salt, the fourth compound is Sn(IV) chlorine e6 lycine amide dihydroxide sodium salt, and the fifth compound is Sn(IV) chlorine e6 lycine amide dihydroxide sodium salt.

    Description

    BRIEF DESCRIPTION OF THE DRAWINGS

    [0025] FIG. 1 illustrates a Lethality curve for ACT4211L.

    [0026] FIG. 2 is a dose-response curve of ACT42111, cytotoxicity of human melanoma cancer cell. X axis represents ACT4211 concentration, Y axis represents the % Cell death when compared to the vehicle control. Each points represents mean+SE (n=6); and FIG. 6 shows the absorption spectrum for ACT4211.

    [0027] FIG. 3 illustrates a Lethality curve for ACT4211.

    [0028] FIG. 4 is a dose-response curve of ACT4211 cytotoxicity of human melanoma cancer cell. X axis represents ACT4211 concentration, Y axis represents the % Cell death when compared to the vehicle control. Each points represents mean+SE (n=6).

    [0029] FIG. 5A represents standard curves used to calculate cell number for each condition wherein y=0.0124X+1620 was used for RFU>5730. X axis represents cell number, Y axis represents RFU measurement.

    [0030] FIG. 5B represents standard curves used to calculate cell number for each condition wherein y=0.022X+397.74 was used for RFU<5730. X axis represents cell number, Y axis represents RFU measurement.

    [0031] FIG. 6 shows the absorption spectrum for ACT4211.

    DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

    [0032] In a some embodiments, the compounds of the present invention are compounds represented by formula (I) as illustrated above, or its geometric isomers, enantiomers, diastereomers, racemates, pharmaceutically acceptable salts, prodrugs and solvates.

    [0033] In some embodiments, the compounds of the present invention are represented by formula (II) as illustrated below, or its geometric isomers, enantiomers, diastereomers, racemates, pharmaceutically acceptable salts, prodrugs and solvates thereof:

    ##STR00002##

    wherein Y is hydroxy, substituted hydroxy, prodrug group or an acceptable metal salt. In some embodiments NR.sub.4R.sub.5 are amino acid, amino acid derivative, or peptide. Amino acid derivatives are those derived from valine, leucine, isoleucine, threonine, methionine, phenylalanine, tryptophan, alanine, arginine, aspartic acid, cysteine, glutamic acid, glycine, histidine, proline, serine, tyrosine, asparagines, and glutamine. Amino acid-like derivatives including, but not limited to taurine, may also be used. Also useful are peptides, particularly those known to have affinity for specific receptors, including but not limited to, oxytocin, vasopressin, bradykinin, LHRH, thrombin, and the like. In some embodiments, NR.sub.4R.sub.5 represents an amine terminated polyethylene glycol (PEG). PEGylation provides for improved bioavailability, including longer circulation time and slower clearance. In particular, it improves the delivery of injectable proteins as well as other compounds. It can also be used for controlled agent release and optimized pharmacokinetics resulting in sustained duration. In addition, PEGylation may improve the safety profile with lower toxicity, immunogenicity, and antigenicity. It can also provide increased efficacy and decreased dosing frequency. PEGylation also improves agent solubility and stability and reduces susceptibility to proteolysis. The PEGs are selected from a broad range of molecular weights (5-60 kDa), functional groups, and attachment chemistries. For example, PEG can be 12-40 kDa, and can be branched or unbranched, binding can be through an NHS reactive group, and binding sites can include lysine or histidine residues. In one example R.sub.4 is hydrogen and R.sub.5 is a (CH.sub.2CH.sub.2O).sub.rCH.sub.2CH.sub.2OH wherein r is an integer between 1 and 100.

    [0034] In some embodiments, the compounds of the present invention are compounds represented by formula (III) as illustrated below, or its geometric isomers, enantiomers, diastereomers, racemates, pharmaceutically acceptable salts, prodrugs and solvates thereof:

    ##STR00003##

    wherein R.sub.6 is hydrogen, a substituted or unsubstituted, saturated or unsaturated alkyl, substituted or unsubstituted aryl; R.sub.7 and is a hydroxy, substituted hydroxy, amine or substituted amine; M and X are as previously defined. In one embodiment, when M is at oxidation state IV, then m=2; some examples are Ti(IV), Zr(IV), Hf(IV), Sn(IV), Mo(IV), V(IV) and W(IV). When M is at oxidation state V, then m=3; some examples are V(V), Nb(V) and Ta(V). When M is at oxidation state VI, then m=4; some examples are Cr(VI), Mo(VI), W(VI) and Re(VI). When M is at oxidation state VII, then m=5; some examples are Tc(VII) and Re(VII). It is preferable to use metal complex compounds of general formula (I) with metals M at oxidation state IV. Preferably M at oxidation state IV includes Si(IV), Ti(IV), Sn(IV), Zr(IV), Hf(IV), Th(IV), Sn(IV), Mo(IV), V(IV) and W(IV). Sn(IV) are particularly preferred.

    [0035] Compounds according to the invention include compounds of formula IV:

    ##STR00004##

    Wherein M, Y.sub.1, Y.sub.2, and NR.sub.4R.sub.5 are set forth in the Table A below:

    Tables A1-6

    [0036]

    TABLE-US-00001 TABLE A1 Cpd. No. M Y.sub.1 Y.sub.2 NR.sub.4R.sub.5 1 Sn(IV) OH OH NHCH.sub.2COOH 2 Sn(IV) OMe OH NHCH.sub.2COOH 3 Sn(IV) OH OMe NHCH.sub.2COOH 4 Sn(IV) OEt OH NHCH.sub.2COOH 5 Sn(IV) OH OEt NHCH.sub.2COOH 6 Sn(IV) NH.sub.2 OH NHCH.sub.2COOH 7 Sn(IV) OH NH.sub.2 NHCH.sub.2COOH 8 Sn(IV) OH OH NHCHCH.sub.3COOH 9 Sn(IV) OMe OH NHCHCH.sub.3COOH 10 Sn(IV) OH OMe NHCHCH.sub.3COOH 11 Sn(IV) OEt OH NHCHCH.sub.3COOH 12 Sn(IV) OH OEt NHCHCH.sub.3COOH 13 Sn(IV) NH.sub.2 OH NHCHCH.sub.3COOH 14 Sn(IV) OH NH.sub.2 NHCHCH.sub.3COOH 15 Sn(IV) OH OH NHCHC.sub.2H.sub.5COOH 16 Sn(IV) OMe OH NHCHC.sub.2H.sub.5COOH 17 Sn(IV) OH OMe NHCHC.sub.2H.sub.5COOH 18 Sn(IV) OEt OH NHCHC.sub.2H.sub.5COOH 19 Sn(IV) OH OEt NHCHC.sub.2H.sub.5COOH 20 Sn(IV) NH.sub.2 OH NHCHC.sub.2H.sub.5COOH 21 Sn(IV) OH NH.sub.2 NHCHC.sub.2H.sub.5COOH 22 Sn(IV) OH OH NHCH(CHPh)COOH 23 Sn(IV) OMe OH NHCH(CHPh)COOH 24 Sn(IV) OH OMe NHCH(CHPh)COOH 25 Sn(IV) OEt OH NHCH(CHPh)COOH 26 Sn(IV) OH OEt NHCH(CHPh)COOH 27 Sn(IV) NH.sub.2 OH NHCH(CHPh)COOH 28 Sn(IV) OH NH.sub.2 NHCH(CHPh)COOH 29 Sn(IV) OH OH NHCH(CHOH)COOH 30 Sn(IV) OMe OH NHCH(CHOH)COOH 31 Sn(IV) OH OMe NHCH(CHOH)COOH 32 Sn(IV) OEt OH NHCH(CHOH)COOH 33 Sn(IV) OH OEt NHCH(CHOH)COOH 34 Sn(IV) NH2 OH NHCH(CHOH)COOH 35 Sn(IV) OH NH2 NHCH(CHOH)COOH

    TABLE-US-00002 TABLE A2 Cpd. No. M Y.sub.1 Y.sub.2 NR.sub.4R.sub.5 36 Sn(IV) OH OH NHCH(CH.sub.2COOH)COOH 37 Sn(IV) OMe OFT NHCH(CH.sub.2COOH)COOH 38 Sn(IV) OH OMe NHCH(CH.sub.2COOH)COOH 39 Sn(IV) OEt OH NHCH(CH.sub.2COOH)COOH 40 Sn(IV) OH OEt NHCH(CH.sub.2COOH)COOH 41 Sn(IV) NH.sub.2 OH NHCH(CH.sub.2COOH)COOH 42 Sn(IV) OH NH.sub.2 NHCH(CH.sub.2COOH)COOH 43 Sn(IV) OH OH NHCH(CH.sub.2CH.sub.2COOH)COOH 44 Sn(IV) OMe OH NHCH(CH.sub.2CH.sub.2COOH)COOH 45 Sn(IV) OH OMe NHCH(CH.sub.2CH.sub.2COOH)COOH 46 Sn(IV) OEt OH NHCH(CH.sub.2CH.sub.2COOH)COOH 47 Sn(IV) OH OEt NHCH(CH.sub.2CH.sub.2COOH)COOH 48 Sn(IV) NH.sub.2 OH NHCH(CH.sub.2CH.sub.2COOH)COOH 49 Sn(IV) OH NH.sub.2 NHCH(CH.sub.2CH.sub.2COOH)COOH 50 Sn(IV) OH OH HNCH((CH.sub.2).sub.4NH.sub.2)COOH 51 Sn(IV) OMe OH HNCH((CH.sub.2).sub.4NH.sub.2)COOH 52 Sn(IV) OH OMe HNCH((CH.sub.2).sub.4NH.sub.2)COOH 53 Sn(IV) OEt OH HNCH((CH.sub.2).sub.4NH.sub.2)COOH 54 Sn(IV) OH OEt HNCH((CH.sub.2).sub.4NH.sub.2)COOH 55 Sn(IV) NH.sub.2 OH HNCH((CH.sub.2).sub.4NH.sub.2)COOH 56 Sn(IV) OH NH.sub.2 HNCH((CH.sub.2).sub.4NH.sub.2)COOH 57 Sn(IV) OH OH HNCH((CH.sub.2).sub.3NH.sub.2)COOH 58 Sn(IV) OMe OH HNCH((CH.sub.2).sub.3NH.sub.2)COOH 59 Sn(IV) OH OMe HNCH((CH.sub.2).sub.3NH.sub.2)COOH 60 Sn(IV) OEt OH HNCH((CH.sub.2).sub.3NH.sub.2)COOH 61 Sn(IV) OH OEt HNCH((CH.sub.2).sub.3NH.sub.2)COOH 62 Sn(IV) NH.sub.2 OH HNCH((CH.sub.2).sub.3NH.sub.2)COOH 63 Sn(IV) OH NH.sub.2 HNCH((CH.sub.2).sub.3NH.sub.2)COOH 64 Sn(IV) OH OH HNCH((CH.sub.2).sub.3NH(CNH.sub.2)NH)COOH 65 Sn(IV) OMe OH HNCH((CH.sub.2).sub.3NH(CNH.sub.2)NH)COOH 66 Sn(IV) OH OMe HNCH((CH.sub.2).sub.3NH(CNH.sub.2)NH)COOH 67 Sn(IV) OEt OH HNCH((CH.sub.2).sub.3NH(CNH.sub.2)NH)COOH 68 Sn(IV) OH OEt HNCH((CH.sub.2).sub.3NH(CNH.sub.2)NH)COOH 69 Sn(IV) NH.sub.2 OH HNCH((CH.sub.2).sub.3NH(CNH.sub.2)NH)COOH 70 Sn(IV) OH NH.sub.2 HNCH((CH.sub.2).sub.3NH(CNH.sub.2)NH)COOH

    TABLE-US-00003 TABLE A3 Cpd. No. M Y.sub.1 Y.sub.2 NR.sub.4R.sub.5 71 Sn(IV) OH OH HNCH(CH.sub.2CONH.sub.2)COOH 72 Sn(IV) OMe OH HNCH(CH.sub.2CONH.sub.2)COOH 73 Sn(IV) OH OMe HNCH(CH.sub.2CONH.sub.2)COOH 74 Sn(IV) OEt OH HNCH(CH.sub.2CONH.sub.2)COOH 75 Sn(IV) OH OEt HNCH(CH.sub.2CONH.sub.2)COOH 76 Sn(IV) NH.sub.2 OH HNCH(CH.sub.2CONH.sub.2)COOH 77 Sn(IV) OH NH.sub.2 HNCH(CH.sub.2CONH.sub.2)COOH 78 Sn(IV) OH OH HNCH(CH.sub.2CH.sub.2CONH.sub.2)COOH 79 Sn(IV) OMe OH HNCH(CH.sub.2CH.sub.2CONH.sub.2)COOH 80 Sn(IV) OH OMe HNCH(CH.sub.2CH.sub.2CONH.sub.2)COOH 81 Sn(IV) OEt OH HNCH(CH.sub.2CH.sub.2CONH.sub.2)COOH 82 Sn(IV) OH OEt HNCH(CH.sub.2CH.sub.2CONH.sub.2)COOH 83 Sn(IV) NH.sub.2 OH HNCH(CH.sub.2CH.sub.2CONH.sub.2)COOH 84 Sn(IV) OH OH (NHCH.sub.2CO).sub.2OH 85 Sn(IV) OH OH (NHCH.sub.2CO).sub.3OH 86 Sn(IV) OH OH (NHCH.sub.2CO).sub.4OH 87 Sn(IV) OH OH (NHCH2CO).sub.5OH 88 Sn(IV) OH OH (NHCH2CO).sub.6OH 89 Sn(IV) OH OH (NHCHCH.sub.3CO).sub.4OH 90 Sn(IV) OH OH (NHCH(CHOH)CO).sub.4OH 91 Sn(IV) OH OH (NHCH((CH.sub.2).sub.3NH.sub.2)CO).sub.2OH 92 Sn(IV) OH OH (NHCH((CH2).sub.3NH.sub.2)CO).sub.3OH 93 Sn(IV) OH OH (NHCH((CH.sub.2).sub.3NH.sub.2)CO).sub.4OH 94 Sn(IV) OH OH (NHCH(CH.sub.2CONH.sub.2)CO).sub.2OH 95 Sn(IV) OH OH N(histidine) 96 Sn(IV) OH OH N(proline) 97 Sn(IV) OH OH NH(CH.sub.2CH.sub.2O).sub.nOH 98 Sn(IV) OH OH N(folate) 99 Sn(IV) OH OMe NH(CH.sub.2CH.sub.2O).sub.nOH 100 Sn(IV) OMe OH NH(CH.sub.2CH.sub.2O).sub.nOH 101 Ti(IV) OH OH NHCH.sub.2COOH 102 Ti(IV) OMe OH NHCH.sub.2COOH 103 Ti(IV) OH OMe NHCH.sub.2COOH 104 Ti(IV) OEt OH NHCH.sub.2COOH 105 Ti(IV) OH OEt NHCH.sub.2COOH

    TABLE-US-00004 TABLE A4 Cpd. No. M Y.sub.1 Y.sub.2 NR.sub.4R.sub.5 106 Ti(IV) NH.sub.2 OH NHCH.sub.2COOH 107 Ti(IV) OH NH.sub.2 NHCH.sub.2COOH 108 Ti(IV) OH OH NHCHCH.sub.3COOH 109 Ti(IV) OMe OH NHCHCH.sub.3COOH 110 Ti(IV) OH OMe NHCHCH.sub.3COOH 111 Ti(IV) OEt OH NHCHCH.sub.3COOH 112 Ti(IV) OH OEt NHCHCH.sub.3COOH 113 Ti(IV) NH.sub.2 OH NHCHCH.sub.3COOH 114 Ti(IV) OH NH.sub.2 NHCHCH.sub.3COOH 115 Ti(IV) OH OH NHCHC.sub.2H.sub.5COOH 116 Ti(IV) OMe OH NHCHC.sub.2H.sub.5COOH 117 Ti(IV) OH OMe NHCHC.sub.2H.sub.5COOH 118 Ti(IV) OEt OH NHCHC.sub.2H.sub.5COOH 119 Ti(IV) OH OEt NHCHC.sub.2H.sub.5COOH 120 Ti(IV) NH.sub.2 OH NHCHC.sub.2H.sub.5COOH 121 Ti(IV) OH NH.sub.2 NHCHC.sub.2H.sub.5COOH 122 Ti(IV) OH OH NHCH(CHPh)COOH 123 Ti(IV) OMe OH NHCH(CHPh)COOH 124 Ti(IV) OH OMe NHCH(CHPh)COOH 125 Ti(IV) OEt OH NHCH(CHPh)COOH 126 Ti(IV) OH OEt NHCH(CHPh)COOH 127 Ti(IV) NH2 OH NHCH(CHPh)COOH 128 Ti(IV) OH NH.sub.2 NHCH(CHPh)COOH 129 Ti(IV) OH OH NHCH(CHOH)COOH 130 Ti(IV) OMe OH NHCH(CHOH)COOH 131 Ti(IV) OH OMe NHCH(CHOH)COOH 132 Ti(IV) OEt OH NHCH(CHOH)COOH 133 Ti(IV) OH OEt NHCH(CHOH)COOH 134 Ti(IV) NH.sub.2 OH NHCH(CHOH)COOH 135 Ti(IV) OH NH.sub.2 NHCH(CHOH)COOH 136 Ti(IV) OH OH NHCH(CH.sub.2COOH)COOH 137 Ti(IV) OMe OH NHCH(CH.sub.2COOH)COOH 138 Ti(IV) OH OMe NHCH(CH.sub.2COOH)COOH 139 Ti(IV) OEt OH NHCH(CH.sub.2COOH)COOH 140 Ti(IV) OH OEt NHCH(CH.sub.2COOH)COOH

    TABLE-US-00005 TABLE A5 Cpd. No. M Y.sub.1 Y.sub.2 NR.sub.4R.sub.5 141 Ti(IV) NH.sub.2 OH NHCH(CH.sub.2COOH)COOH 142 Ti(IV) OH NH.sub.2 NHCH(CH.sub.2COOH)COOH 143 Ti(IV) OH OH NHCH(CH.sub.2CH.sub.2COOH)COOH 144 Ti(IV) OMe OH NHCH(CH.sub.2CH.sub.2COOH)COOH 145 Ti(IV) OH OMe NHCH(CH.sub.2CH.sub.2COOH)COOH 146 Ti(IV) OEt OH NHCH(CH.sub.2CH.sub.2COOH)COOH 147 Ti(IV) OH OEt NHCH(CH.sub.2CH.sub.2COOH)COOH 148 Ti(IV) NH.sub.2 OH NHCH(CH.sub.2CH.sub.2COOH)COOH 149 Ti(IV) OH NH.sub.2 NHCH(CH.sub.2CH.sub.2COOH)COOH 150 Ti(IV) OH OH HNCH((CH.sub.2).sub.4NH.sub.2)COOH 151 Ti(IV) OMe OH HNCH((CH.sub.2).sub.4NH.sub.2)COOH 152 Ti(IV) OH OMe HNCH((CH.sub.2).sub.4NH.sub.2)COOH 153 Ti(IV) OEt OH HNCH((CH.sub.2).sub.4NH.sub.2)COOH 154 Ti(IV) OH OEt HNCH((CH.sub.2).sub.4NH.sub.2)COOH 155 Ti(IV) NH.sub.2 OH HNCH((CH.sub.2).sub.4NH.sub.2)COOH 156 Ti(IV) OH NH.sub.2 HNCH((CH.sub.2).sub.4NH.sub.2)COOH 157 Ti(IV) OH OH HNCH((CH.sub.2).sub.3NH.sub.2)COOH 158 Ti(IV) OMe OH HNCH((CH.sub.2).sub.3NH.sub.2)COOH 159 Ti(IV) OH OMe HNCH((CH.sub.2).sub.3NH.sub.2)COOH 160 Ti(IV) OEt OH HNCH((CH.sub.2).sub.3NH.sub.2)COOH 161 Ti(IV) OH OEt HNCH((CH.sub.2).sub.3NH.sub.2)COOH 162 Ti(IV) NH.sub.2 OH HNCH((CH.sub.2).sub.3NH.sub.2)COOH 163 Ti(IV) OH NH.sub.2 HNCH((CH.sub.2).sub.3NH.sub.2)COOH 164 Ti(IV) OH OH HNCH((CH.sub.2).sub.3NH(CNH.sub.2)NH)COOH 165 Ti(IV) OMe OH HNCH((CH.sub.2).sub.3NH(CNH.sub.2)NH)COOH 166 Ti(IV) OH OMe HNCH((CH.sub.2).sub.3NH(CNH.sub.2)NH)COOH 167 Ti(IV) OEt OH HNCH((CH.sub.2).sub.3NH(CNH.sub.2)NH)COOH 168 Ti(IV) OH OEt HNCH((CH.sub.2).sub.3NH(CNH.sub.2)NH)COOH 169 Ti(IV) NH.sub.2 OH HNCH((CH.sub.2).sub.3NH(CNH.sub.2)NH)COOH 170 Ti(IV) OH NH.sub.2 HNCH((CH.sub.2).sub.3NH(CNH.sub.2)NH)COOH 171 Ti(IV) OH OH HNCH(CH.sub.2CONH.sub.2)COOH 172 Ti(IV) OMe OH HNCH(CH.sub.2CONH.sub.2)COOH 173 Ti(IV) OH OMe HNCH(CH.sub.2CONH.sub.2)COOH 174 Ti(IV) OEt OH HNCH(CH.sub.2CONH.sub.2)COOH 175 Ti(IV) OH OEt HNCH(CH.sub.2CONH.sub.2)COOH

    TABLE-US-00006 TABLE A6 Cpd. No. M Y.sub.1 Y.sub.2 NR.sub.4R.sub.5 176 Ti(IV) NH.sub.2 OH HNCH(CH.sub.2CONH.sub.2)COOH 177 Ti(IV) OH NH.sub.2 HNCH(CH.sub.2CONH.sub.2)COOH 178 Ti(IV) OH OH HNCH(CH.sub.2CH.sub.2CONH.sub.2)COOH 179 Ti(IV) OMe OH HNCH(CH.sub.2CH.sub.2CONH.sub.2)COOH 180 Ti(IV) OH OMe HNCH(CH.sub.2CH.sub.2CONH.sub.2)COOH 181 Ti(IV) OEt OH HNCH(CH.sub.2CH.sub.2CONH.sub.2)COOH 182 Ti(IV) OH OEt HNCH(CH.sub.2CH.sub.2CONH.sub.2)COOH 183 Ti(IV) NH.sub.2 OH HNCH(CH.sub.2CH.sub.2CONH.sub.2)COOH 184 Ti(IV) OH OH (NHCH.sub.2CO).sub.2OH 185 Ti(IV) OH OH (NHCH.sub.2CO).sub.3OH 186 Ti(IV) OH OH (NHCH.sub.2CO).sub.4OH 187 Ti(IV) OH OH (NHCH.sub.2CO).sub.5OH 188 Ti(IV) OH OH (NHCH.sub.2CO).sub.6OH 189 Ti(IV) OH OH (NHCHCH.sub.3CO).sub.4OH 190 Ti(IV) OH OH (NHCH(CHOH)CO).sub.4OH 191 Ti(IV) OH OH (NHCH((CH.sub.2).sub.3NH.sub.2)CO).sub.2OH 192 Ti(IV) OH OH (NHCH((CH.sub.2).sub.3NH.sub.2)CO).sub.3OH 193 Ti(IV) OH OH (NHCH((CH.sub.2).sub.3NH.sub.2)CO).sub.4OH 194 Ti(IV) OH OH (NHCH(CH.sub.2CONH.sub.2)CO).sub.2OH 195 Ti(IV) OH OH N(histidine) 196 Ti(IV) OH OH N(proline) 197 Ti(IV) OH OH NH(CH.sub.2CH.sub.2O).sub.nOH 198 Ti(IV) OH OH N(folate) 199 Ti(IV) OH OMe NH(CH.sub.2CH.sub.2O).sub.nOH 200 Ti(IV) OMe OH NH(CH.sub.2CH.sub.2O).sub.nOH

    [0037] Compounds according to the invention include compounds of formula:

    ##STR00005##

    wherein M, Y.sub.1, Y.sub.2, and R.sub.4 are set forth in the Table (B) below:

    TABLE-US-00007 TABLE B Cpd. No. M Y.sub.1 Y.sub.2 R.sub.4 1 Sn(IV) OH OH H 2 Sn(IV) OMe OH H 3 Sn(IV) OH OMe H 4 Sn(IV) OEt OH H 5 Sn(IV) OH OEt H 6 Sn(IV) NH.sub.2 OH H 7 Sn(IV) OH NH.sub.2 H 8 Sn(IV) OH OH CH.sub.3 9 Sn(IV) OMe OH CH.sub.3 10 Sn(IV) OH OMe CH.sub.3 11 Sn(IV) OEt OH CH.sub.3 12 Sn(IV) OH OEt CH.sub.3 13 Sn(IV) NH.sub.2 OH CH.sub.3 14 Sn(IV) OH NH.sub.2 CH.sub.3 15 Sn(IV) OH OH C.sub.2H.sub.5 16 Sn(IV) OMe OH C.sub.2H.sub.5 17 Sn(IV) OH OMe C.sub.2H.sub.5 18 Sn(IV) OEt OH C.sub.2H.sub.5 19 Sn(IV) OH OEt C.sub.2H.sub.5 20 Sn(IV) NH.sub.2 OH C.sub.2H.sub.5 21 Sn(IV) OH NH.sub.2 C.sub.2H.sub.5 22 Sn(IV) OH OH CH.sub.2Ph 23 Sn(IV) OMe OH CH.sub.2Ph 24 Sn(IV) OH OMe CH.sub.2Ph 25 Sn(IV) OEt OH CH.sub.2Ph 26 Sn(IV) OH OEt CH.sub.2Ph 27 Sn(IV) NH2 OH CH.sub.2Ph 28 Sn(IV) OH NH.sub.2 CH.sub.2Ph

    [0038] In one aspect an agent for use in treatment of a disease state or improving a condition associated with a disease state, the agent comprises one or more components selected from the group consisting of Sn(IV) chlorin e6 gly-gly amide dihydroxide sodium salt, Sn(IV) chlorin e6 gly-gly-gly-gly amide dihydroxide sodium salt, Sn(IV) chlorin e6 Taurine amide dihydroxide sodium salt, Sn(IV) chlorin e6 L-serine amide dihydroxide sodium salt, Sn(IV) chlorin e6 lycine amide dihydroxide sodium salt. In some embodiments two components are combined in a weight ratio between 10:1 and 1:10. In some embodiments three components are combined in a weight ratio between 10:1:1, 10:10:1, 1:10:1, 1:10:10 and 1:1:10. In another aspect an agent for use in treatment of a disease state or improving a condition associated with the disease, the agent comprising four components, the four components combined such that the greatest is present at not more than 70% by weight and the least is present at not less than 10% by weight. In some embodiments, the disease state is cancer, including tumors.

    [0039] In some embodiments, the four components are selected from the group consisting of Sn(IV) chlorin c6 gly-gly amide dihydroxide sodium salt, Sn(IV) chlorin e6 gly-gly-gly-gly amide dihydroxide sodium salt, Sn(IV) chlorin e6 Taurine amide dihydroxide sodium salt, Sn(IV) chlorin e6 L-serine amide dihydroxide sodium salt. In some embodiments, the first component is Sn(IV) chlorin e6 gly-gly amide dihydroxide sodium salt, the second component is Sn(IV) chlorin e6 gly-gly-gly-gly amide dihydroxide sodium salt, the third component is Sn(IV) chlorin e6 Taurine amide dihydroxide sodium salt, the forth component is Sn(IV) chlorin e6 L-serine amide dihydroxide sodium salt.

    [0040] In some embodiments, the four components are selected from the group consisting of Sn(IV) chlorin e6 gly-gly amide dihydroxide sodium salt, Sn(IV) chlorin e6 gly-gly-gly-gly amide dihydroxide sodium salt, Sn(IV) chlorin e6 Taurine amide dihydroxide sodium salt, Sn(IV) chlorin e6 L-serine amide dihydroxide sodium salt. In some embodiments, the first component is Sn(IV) chlorin e6 gly-gly amide dihydroxide sodium salt, the second component is Sn(IV) chlorin e6 gly-gly-gly-gly amide dihydroxide sodium salt, the third component is Sn(IV) chlorin e6 Taurine amide dihydroxide sodium salt, the forth component is Sn(IV) chlorin c6 lycine amide dihydroxide sodium salt.

    [0041] In another aspect an agent for use in treatment of a disease state or improving a condition associated with the disease, the agent comprising five components, the five components arc combined such that the greatest is present at not more than 60% by weight and the least is present at not less than 10% by weight.

    [0042] In some embodiments, the five components are selected from the group consisting of Sn(IV) chlorin e6 gly-gly amide dihydroxide sodium salt, Sn(IV) chlorin e6 gly-gly-gly gly amide dihydroxide sodium salt, Sn(IV) chlorin e6 Taurine amide dihydroxide sodium salt, Sn(IV) chlorin e6 L-serine amide dihydroxide sodium salt, Sn(IV) chlorin e6 lycine amide dihydroxide sodium salt. In some embodiments, the first component is Sn(IV) chlorin e6 gly-gly amide dihydroxide sodium salt, the second component is Sn(IV) chlorin e6 gly-gly-gly-gly amide dihydroxide sodium salt, the third component is Sn(IV) chlorin e6 Taurine amide dihydroxide sodium salt, the forth component is Sn(IV) chlorin e6 L-serine amide dihydroxide sodium salt, and the fifth component is Sn(IV) chlorin e6 lycine amide dihydroxide sodium salt.

    [0043] The invention also provides a method for improving a condition associated with a disease state, using energy-activated therapy whereby the energy source is either ultrasound, light, or a combination of ultrasound or light, the method comprising administering an amount of an agent according to the invention to a mammal effective to improve one or more conditions of the mammal associated with a disease state or disease states.

    [0044] In some embodiments the agent has a first component, a second component, a third component, a fourth component, and a fifth component in weight ratios such that the component present in the highest amount is present at 20-96% and the component present at the lowest amount is present at 1-20%. In some embodiments, the first component is Sn(IV) chlorin e6 gly-gly amide dihydroxide sodium salt, the second component is Sn(IV) chlorin e6 gly-gly-gly-gly amide dihydroxide sodium salt, the third component is Sn(IV) chlorin e6 Taurine amide dihydroxide sodium salt, the fourth component is Sn(IV) chlorin e6 L-serine amide dihydroxide sodium salt, and the fifth component is Sn(IV) chlorin e6 lycine amide dihydroxide sodium salt. In some embodiments the components are ground together to a soluble powder and administered to the mammal in that form. In some embodiments the components are dissolved in water and administered to the mammal in that form. In some embodiments the components are lyophilized to form an active ingredient part of the agent. In some embodiments the disease is cancer, including tumors. In some embodiments, the agent comprises a sonosensitizer. In some embodiments the agent comprises a photosensitizer. In some embodiments the agent comprises both a photosensitizer and a sonosensitizer.

    [0045] Photodynamic therapy and sonodynamic therapy use agents to treat or ameliorate conditions associated with one or more disease states. Diseases states may include, for example, one or more types of cancer, including tumors. Particular agents that may be used include multiple component metal complexes. In some embodiments the metal complexes include tin. Some embodiments include chlorin e6. Some embodiments include aspartyl chlorin e6. In some embodiments a first metal complex is Sn(IV) chlorin e6 gly-gly amide dihydroxide sodium salt. In some embodiments, the second metal complex is Sn(IV) chlorin e6 gly-gly-gly-gly amide dihydroxide sodium salt. In some embodiments, the third metal complex is Sn(IV) chlorin e6 Taurine amide dihydroxide sodium salt. In some embodiments, the forth metal complex is Sn(IV) chlorin e6 L-serine amide dihydroxide sodium salt. In some embodiments first, second, third, and fourth metal complexes are combined into an agent in a weight ratio of approximately 4:2:1:1. Herein the term approximately includes values +/2-10%.

    [0046] In some embodiments a first metal complex is Sn(IV) chlorin e6 gly-gly amide dihydroxide sodium salt. In some embodiments, the second metal complex is Sn(IV) chlorin e6 gly-gly-gly-gly amide dihydroxide sodium salt. In some embodiments, the third metal complex is Sn(IV) chlorin e6 Taurine amide dihydroxide sodium salt. In some embodiments, the forth metal complex is Sn(IV) chlorin e6 lycine amide dihydroxide sodium salt. In some embodiments first, second, third, and fourth metal complexes are combined into an agent in a weight ratio of approximately 4:2:1:1. Herein the term approximately includes values +/2-10%.

    [0047] In some embodiments an agent for use in treatment of a disease state or improving a condition associated with the disease state, comprises one or more components selected from the group consisting of Sn(IV) chlorin e6 gly-gly amide dihydroxide sodium salt, Sn(IV) chlorin e6 gly-gly-gly-gly amide dihydroxide sodium salt, Sn(IV) chlorin e6 Taurine amide dihydroxide sodium salt, Sn(IV) chlorin e6 L-serine amide dihydroxide sodium salt, Sn(IV) chlorin e6 lycine amide dihydroxide sodium salt. In some embodiments comprising five components, each of the five components is combined in the agent in a weight ratio relation to the other four components between approximately 10:0:0:0:0 to approximately 0:10:10:10:10.

    [0048] For example, one active composition comprising the following chemical components in the following weight ratios has been found to be effective in the treatment of various diseases in the body using ultrasound and/or light therapy. Each component is a good photodynamic agent. This is referred to in the Examples as ACT4211.

    TABLE-US-00008 Weight Component Ratio Sn(IV) chlorin e6 gly-gly amide dihydroxide sodium salt 4 Sn(IV) chlorin e6 gly-gly-gly-gly amide dihydroxide sodium salt 2 Sn(IV) chlorin e6 Taurine amide dihydroxide sodium salt 1 Sn (1V) chlorin e6 L-serine amide dihydroxide sodium salt 1

    [0049] In another example, an active composition comprising the following chemical components in the following weight ratios has been prepared and is to be tested in the treatment of various diseases in the body using ultrasound and/or light therapy. Each component is a good photodynamic agent. This is referred to in the Examples as ACT4211L.

    TABLE-US-00009 Weight Component Ratio Sn(IV) chlorin e6 gly-gly amide dihydroxide sodium salt 4 Sn(IV) chlorin e6 gly-gly-gly-gly amide dihydroxide sodium salt 2 Sn(IV) chlorin e6 Taurine amide dihydroxide sodium salt 1 Sn (1V) chlorin e6 lycine amide dihydroxide sodium salt 1

    [0050] In another example, an active composition comprising the following chemical components in the following weight ratios has been prepared to be tested in the treatment of various diseases in the body using ultrasound and/or light therapy. Each component is a good photodynamic agent.

    TABLE-US-00010 Weight Component Ratio Sn(IV) chlorin e6 gly-gly amide dihydroxide sodium salt 4 Sn(IV) chlorin e6 gly-gly-gly-gly amide dihydroxide sodium salt 2 Sn(IV) chlorin e6 Taurine amide dihydroxide sodium salt 1 Sn (1V) chlorin e6 L-serine amide dihydroxide sodium salt 1 Sn (1V) chlorin e6 lycine amide dihydroxide sodium salt 1

    [0051] There are discussions in the art of various types of metal complexes. For example, U.S. Pat. No. 4,656,186 mentions a serine free base compound, but not a tin chelate. U.S. Pat. Nos. 4,656,186, 4,675,338, 4,693,885; 4,977,177; 5,004,811; and 5,066,274 similarly mention preparation of metal complexes, but do not mention the tin chelates. U.S. Pat. No. 4,977,177 teaches attaching more than one amino acid to the tetrapyrrole at several sites. Each of the above references is herby incorporated by reference in its entirety.

    [0052] In another aspect, the present invention provides a pharmaceutical composition comprising a compound of the invention and a pharmaceutically acceptable carrier.

    [0053] The compounds of the present invention are formulated into final pharmaceutical compositions for administration to a subject or applied to an in vitro target using techniques well-known in the art, for example, as summarized in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. The compounds of the invention are useful as photosensitizers, as sonosensitizers as therapeutic, and diagnostic agents, for example for treatment of several cancer types such as, but not limited to, melanoma, prostate, brain, colon, ovarian, breast, skin, lung, esophagus and bladder cancers and other hormone-sensitive tumors, as well as for treatment of age-related macular degeneration, and for killing cells, viruses, fungi and bacteria in samples and living tissues as well known in the art of PDT and other sonosensitizer applications.

    [0054] The compounds of the invention arc useful, for example, in sensitizing neoplastic cells or other abnormal tissue to destruction by ultrasound frequencies. The wavelength of the ultrasound is preferably chosen to match the maximum absorbance of the sonosensitizer. The suitable energy for any of the compounds can readily be determined empirically but can be between 20 KHz to 20 MHz, intensity of 0.1 to 500 W/cm2 and duration of 0.5 sec. to 5 hours. In an alternative embodiment, the compounds can be activated by light waves, as typically employed in photodynamic therapy. In an alternative embodiment, the compounds can be activated by a combination of light waves and ultrasound.

    [0055] The conjugation of proteins, e.g., hormones, growth factors or their derivatives, antibodies, peptides that bind specifically to target cells receptors, and of cell nutrients, e.g. tyrosine, can increase their retention in tumor and treated sites.

    [0056] The invention further relates to a method of sonodynamic therapy, which comprises administering to a subject a therapeutically effective amount of a compound of the invention, followed by local ultrasound.

    [0057] The compounds of the invention are also useful for sonodestruction of normal or malignant animal cells, as desired. Thus, the invention further provides the use of the compounds of the invention for in vivo, ex-vivo or in vitro killing of cells or infectious agents such as bacteria, viruses, parasites and fungi in a biological product, e.g. blood, Use of the compounds accordingly comprises treating the infected sample with the compound followed by ultrasound and/or red light irradiation of the sample. The present invention provides for the use of one or more compounds of the invention in the manufacture of a medicament for the treatment of cancer.

    [0058] In some embodiments, the present invention includes the use of one or more compounds of the invention in the manufacture of a medicament that prevents further aberrant proliferation, differentiation, or survival of cells. For example, compounds of the invention may be useful in preventing tumors from increasing in size or from reaching a metastatic state. The subject compounds may be administered to halt or inhibit the progression or advancement of cancer or to induce tumor necrosis, tumor apoptosis, or inhibit tumor angiogenesis.

    [0059] In addition, the instant invention includes use of the subject compounds to prevent a recurrence of cancer. This is accomplished in part because the use of the compounds in a therapeutic mode creates an inflammatory reaction and/or response that yields a vaccine effect.

    [0060] This invention further embraces the treatment or prevention of cell proliferative disorders such as hyperplasias, dysplasias and pre-cancerous lesions. Dysplasia is the earliest form of pre-cancerous lesion recognizable in a biopsy by a pathologist. The subject compounds may be administered for the purpose of preventing said hyperplasias, dysplasias or pre-cancerous lesions from continuing to expand or from becoming cancerous. Examples of pre-cancerous lesions may occur in skin, esophageal tissue, breast and cervical intra-epithelial tissue.

    [0061] Combination therapy includes the administration of the subject compounds in further combination with other biologically active ingredients (such as, but not limited to, a second and different antineoplastic agent) and non-drug therapies (such as, but not limited to, surgery or radiation treatment). For instance, the compounds of the invention can be used in combination with other pharmaceutically active compounds, preferably compounds that are able to enhance the effect of the compounds of the invention. The compounds of the invention can be administered simultaneously (as a single preparation or separate preparation) or sequentially to the other drug therapy. In general, a combination therapy envisions administration of two or more drugs during a single cycle or course of therapy.

    [0062] Alternatively or additionally, administration of the subject compounds can be staggered, thereby resulting in varied compartmental distribution.

    [0063] The compounds are administered prior to activation by ultrasound or phototherapy. Preferably the compounds are administered at least one day before activation, generally between 2 and 5 days before activation, or between 24 and 96 hours before activation. In one aspect of the invention, the subject compounds may be administered in combination with one or more separate agents that modulate protein kinases involved in various disease states. Examples of such kinases may include, but are not limited to: serine/threonine specific kinases, receptor tyrosine specific kinases and non-receptor tyrosine specific kinases. Serine/threonine kinases include mitogen activated protein kinases (MAPK), meiosis specific kinase (Aurora), RAF and Aurora kinase. Examples of receptor kinase families include epidermal growth factor receptor (EGFR) (e.g. HER2/neu, HER3, HER4, ErbB, ErbB2, ErbB3, ErbB4, Xmrk, DER, Let23); fibroblast growth factor (FGF) receptor (e.g. FGF-R1, GFF-R2/BEK/CEK3, FGF-R3/CEK2, FGF-R4/TKF, KGF R); hepatocyte growth/scatter factor receptor (HGFR) (e.g, MET, RON, SEA, SEX); insulin receptor (e.g. IGFI-R); Eph (e.g. CEK5, CEK8, EBK, ECK, EEK, EHK-1, EHK-2, ELK, EPH, ERK, HEK, MDK2, MDK5, SEK); Axl (e.g. Mer/Nyk, Rse); RET; and platelet-derived growth factor receptor (PDGFR) (e.g. PDGF-R, PDG-R, CSF1-R/FMS, SCF-R/C-KIT, VEGF-R/FLT, NEK/FLK1, FLT3/FLK2/STK-1). Non-receptor tyrosine kinase families include, but are not limited to, BCR-ABL (e.g. p43abl, ARG); BTK (e.g. ITK/EMT, TEC); CSK, FAK, FPS, JAK, SRC, BMX, FER, CDK and SYK.

    [0064] In certain embodiments, the compounds of the invention are administered in combination with a known chemotherapeutic agent.

    [0065] In certain embodiments, the compounds of the invention are administered in combination with a chemoprotective agent. Chemoprotective agents act to protect the body or minimize the side effects of chemotherapy. Examples of such agents include, but are not limited to, amfostine, mesna, and dexrazoxane.

    [0066] In some embodiments of the invention, the subject compounds are administered in combination with radiation therapy. Radiation is commonly delivered internally (implantation of radioactive material near cancer site) or externally from a machine that employs photon (x-ray or gamma-ray) or particle radiation. Where the combination therapy further comprises radiation treatment, the radiation treatment may be conducted at any suitable time so long as a beneficial effect from the co-action of the combination of the therapeutic agents and radiation treatment is achieved. For example, in appropriate cases, the beneficial effect is still achieved when the radiation treatment is temporally removed from the administration of the therapeutic agents, perhaps by days or even weeks.

    [0067] It will be appreciated that compounds of the invention can be used in combination with an immunotherapeutic agent. One form of immunotherapy is the generation of an active systemic tumor-specific immune response of host origin by administering a vaccine composition at a site distant from the tumor. Various types of vaccines have been proposed, including isolated tumor-antigen vaccines and anti-idiotype vaccines. Another approach is to use tumor cells from the subject to be treated, or a derivative of such cells (reviewed by Schirrmacher et al. (1995) J. Cancer Res. Clin. Oncol. 121:487). In U.S. Pat. No. 5,484,596, Hanna Jr. et al. claims a method for treating a resectable carcinoma to prevent recurrence or metastases, comprising surgically removing the tumor, dispersing the cells with collagenase, irradiating the cells, and vaccinating the patient with at least three consecutive doses of about 10.sup.7 cells.

    [0068] It will be appreciated that the compounds of the invention may advantageously be used in conjunction with one or more known adjunctive therapeutic agents.

    [0069] In some embodiments, compounds of the invention can be used to induce or inhibit apoptosis, a physiological cell death process critical for normal development and homeostasis. Alterations of apoptotic pathways contribute to the pathogenesis of a variety of human diseases. Compounds of the invention, as modulators of apoptosis, will be useful in the treatment of a variety of human diseases with aberrations in apoptosis including cancer (particularly, but not limited to, follicular lymphomas, carcinomas with p53 mutations, hormone dependent tumors of the breast, prostate and ovary, and precancerous lesions such as familial adenomatous polyposis).

    DEFINITIONS

    [0070] Listed below are definitions of various terms used to describe this invention. Unless a specific meaning is stated for a term used herein, it is intended that the term be given its usual meaning in the art.

    [0071] The term substituted refers to the replacement of one or more hydrogen radicals in a given structure with the radical of a specified substituent including, but not limited to: halo, alkyl, alkenyl, alkynyl, aryl, heterocyclyl, thiol, alkylthio, arylthio, alkylthioalkyl, arylthioalkyl, alkylsulfonyl, alkylsulfonylalkyl, arylsulfonylalkyl, alkoxy, aryloxy, aralkoxy, aminocarbonyl, aminocarbonylcycloalkyl, aminocarbonylheterocyclyl, alkylaminocarbonyl, arylaminocarbonyl, alkoxycarbonyl, aryloxycarbonyl, haloalkyl, amino, trifluoromethyl, cyano, nitro, alkylamino, arylamino, alkylaminoalkyl, arylaminoalkyl, aminoalkylamino, hydroxy, alkoxyalkyl, carboxyalkyl, alkoxycarbonylalkyl, aminocarbonylalkyl, acyl, aralkoxycarbonyl, carboxylic acid sulfonic acid, sulfonyl, phosphonic acid, aryl, heteroaryl, heterocyclic, and aliphatic. It is understood that the substituent may be further substituted.

    [0072] For simplicity, chemical moieties are defined and referred to throughout can be univalent chemical moieties (e.g., alkyl, aryl, etc.) or multivalent moieties under the appropriate structural circumstances clear to those skilled in the art. For example, an alkyl moiety can be referred to a monovalent radical (e.g. CH.sub.3CH.sub.2), or in other instances, a bivalent linking moiety can be alkyl, in which case those skilled in the art will understand the alkyl to be a divalent radical (e.g., CH.sub.2CH.sub.2), which is equivalent to the term alkylene.

    [0073] The phrase adjunctive therapy encompasses treatment of a subject with agents that reduce or avoid side effects associated with the combination therapy of the present invention, including, but not limited to, those agents, for example, that reduce the toxic effect of anticancer drugs, e.g., bone resorption inhibitors, cardioprotective agents; prevent or reduce the incidence of nausea and vomiting associated with chemotherapy, radiotherapy or surgical procedures; or reduce the incidence of infection associated with the administration of myelosuppressive anticancer drugs.

    [0074] The term effective amount of the subject compounds, with respect to the subject method of treatment, refers to an amount of the subject compound which, when delivered as part of desired dose regimen, brings about, e.g. a change in the rate of cell proliferation and/or state of differentiation and/or rate of survival of a cell to clinically acceptable standards. This amount may further relieve to some extent one or more of the symptoms of a neoplasia disorder, including, but is not limited to: 1) reduction in the number of cancer cells; 2) reduction in tumor size; 3) inhibition (i.e., slowing to some extent, preferably stopping) of cancer cell infiltration into peripheral organs; 4) inhibition (i.e., slowing to some extent, preferably stopping) of tumor metastasis; 5) inhibition, to some extent, of tumor growth; 6) relieving or reducing to some extent one or more of the symptoms associated with the disorder; and/or 7) relieving or reducing the side effects associated with the administration of anticancer agents.

    [0075] The term inhibition, in the context of neoplasia, tumor growth or tumor cell growth, may be assessed by delayed appearance of primary or secondary tumors, slowed development of primary or secondary tumors, decreased occurrence of primary or secondary tumors, slowed or decreased severity of secondary effects of disease, arrested tumor growth and regression of tumors, among others. In the extreme, complete inhibition, is referred to herein as prevention or chemoprevention.

    [0076] The phrase a radio therapeutic agent refers to the use of electromagnetic or particulate radiation in the treatment of neoplasia.

    [0077] The term recurrence as used herein refers to the return of cancer after a period of remission. This may be due to incomplete removal of cells from the initial cancer and may occur locally (the same site of initial cancer), regionally (in vicinity of initial cancer, possibly in the lymph nodes or tissue), and/or distally as a result of metastasis.

    [0078] The term treatment refers to any process, action, application, therapy, or the like, wherein a mammal, including a human being, is subject to medical aid with the object of improving the mammal's condition, directly or indirectly.

    [0079] By a therapeutically effective amount of a compound of the invention is meant an amount of the compound that confers a therapeutic effect on the treated subject, at a reasonable benefit/risk ratio applicable to any medical treatment. The therapeutic effect may be objective (i.e., measurable by some test or marker) or subjective (i.e., subject gives an indication of or feels an effect). An effective amount of the compound described above may range from about 0.01 mg/Kg to about 500 mg/Kg, preferably from about 0.1 to about 10 mg/Kg. Effective doses will also vary depending on route of administration, as well as the possibility of co-usage with other agents. It will be understood, however, that the total daily usage of the compounds and compositions of the present invention will be decided by the attending physician within the scope of sound medical judgment. The specific therapeutically effective dose level for any particular patient will depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific compound employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and rate of excretion of the specific compound employed; the duration of the treatment; drugs used in combination or contemporaneously with the specific compound employed; and like factors well known in the medical arts.

    [0080] As used herein, the term pharmaceutically acceptable salt refers to those salts which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response and the like, and are commensurate with a reasonable benefit/risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, S. M. Berge, et al. describes pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66: 1-19 (1977). As used herein, the term pharmaceutically acceptable ester refers to esters that hydrolyze in vivo and include those that break down readily in the human body to leave the parent compound or a salt thereof. Suitable ester groups include, for example, those derived from pharmaceutically acceptable aliphatic carboxylic acids.

    [0081] The term pharmaceutically acceptable prodrugs as used herein refers to those prodrugs of the compounds of the present invention which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and lower animals with undue toxicity, irritation, allergic response, and the like, commensurate with a reasonable benefit/risk ratio, and effective for their intended use, as well as the zwitterionic forms, where possible, of the compounds of the present invention. Prodrug, as used herein means a compound that is convertible in vivo by metabolic means (e.g. by hydrolysis) to a compound of the invention. Various forms of prodrugs are known in the art, for example, as discussed in Bundgaard, (ed.), Design of Prodrugs, Elsevier (1985); Widder, et al. (ed.), Methods in Enzymology, vol. 4, Academic Press (1985); Krogsgaardcustom-characterLarsen, et al., (ed). Design and Application of Prodrugs, Textbook of Drug Design and Development, Chapter 5, 113-191 (1991); Bundgaard, et al., Journal of Drug Deliver Reviews, 8:1-38(1992); Bundgaard, J. of Pharmaceutical Sciences, 77:285 et seq. (1988); Higuchi and Stella (eds.) Prodrugs as Novel Drug Delivery Systems, American Chemical Society (1975); and Bernard Testa & Joachim Mayer, Hydrolysis In Drug And Prodrug Metabolism: Chemistry, Biochemistry And Enzymology, John Wiley and Sons, Ltd. (2002).

    [0082] The term, pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration, such as sterile pyrogen-free water. Suitable carriers arc described in the most recent edition of Remington's Pharmaceutical Sciences, a standard reference text in the field, which is incorporated herein by reference. The term subject as used herein refers to an animal. Preferably the animal is a mammal. More preferably the mammal is a human. A subject also refers to, for example, dogs, cats, horses, cows, pigs, guinea pigs, fish, birds and the like.

    [0083] The compounds of this invention may be modified by appending appropriate functional groups and/or moieties to enhance selective biological properties. Such modifications are known in the art and may include those which increase biological penetration into a given biological system (e.g., blood, lymphatic system, central nervous system), increase oral availability, increase solubility to allow administration by injection, alter metabolism and alter rate of excretion.

    [0084] The compounds described herein contain one or more asymmetric centers and thus give rise to enantiomers, diastereomers, and other stereoisomeric forms that may be defined, in terms of absolute stereochemistry, as (R)- or (S)-, or as (D)- or (L)- for amino acids. The present invention is meant to include all such possible isomers, as well as their racemic and optically pure forms. Optical isomers may be prepared from their respective optically active precursors by the procedures described above, or by resolving the racemic mixtures. The resolution can be carried out in the presence of a resolving agent, by chromatography or by repeated crystallization or by some combination of these techniques that are known to those skilled in the art. Further details regarding resolutions can be found in Jacques, et al., Enantiomers, Racemates, and Resolutions (John Wiley & Sons, 1981). When the compounds described herein contain olefinic double bonds, other unsaturation, or other centers of geometric asymmetry, and unless specified otherwise, it is intended that the compounds include both E and Z geometric isomers and/or cis- and trans-isomers. Likewise, all tautomeric forms are also intended to be included. The configuration of any carbon-carbon double bond appearing herein is selected for convenience only and is not intended to designate a particular configuration unless the text so states; thus a carbon-carbon double bond or carbon-heteroatom double bond depicted arbitrarily herein as trans may be cis, trans, or a mixture of the two in any proportion.

    Pharmaceutical Compositions

    [0085] The pharmaceutical compositions of the present invention comprise a therapeutically effective amount of a compound or compounds. In some embodiments, the active ingredients and mixtures of active ingredients may be used, for example, in pharmaceutical compositions comprising a pharmaceutically acceptable carrier prepared for storage and subsequent administration. Also, some embodiments include use of the above-described active ingredients with a pharmaceutically acceptable carrier or diluent. Acceptable carriers or diluents for therapeutic use are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, 18th Ed., Mack Publishing Co., Easton, PA (1990).

    Methods of Administration

    [0086] The pharmaceutical compositions of this invention may be administered sublingually, orally, parenterally, by inhalation spray, topically, rectally, nasally, buccally, vaginally or via an implanted reservoir, preferably by oral administration or administration by injection, as deemed appropriate by those of skill in the art for bringing the compositions of the invention into optimal contact with the target tissue.

    EXAMPLES

    [0087] The compounds of the present invention will be better understood in connection with the following non-limiting examples, which are intended as an illustration only and not limiting of the scope of the invention. Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art and such changes and modifications including, without limitation, those relating to the chemical structures, substituents, derivatives, formulations and/or methods of the invention may be made without departing from the spirit of the invention and the scope of the claims. Compounds of the invention can be prepared by procedures known to those skilled in the art, such as U.S. Pat. No. 6,462,192 that is hereby incorporated by reference by its entirety.

    Example 1. Treatment of Skin Cancer

    [0088] As an example, consider photodynamic therapy as a treatment for basal cell carcinoma. Basal cell carcinoma is the most common form of skin cancer in humans. Conventional treatment of basal cell carcinoma involves surgical excision, cryogenic treatment with liquid nitrogen, or localized chemotherapy with 5-fluorouracil or other agents. Applying a photosensitizer precursor (aminolevulinic acid or methyl aminolevulinate). A waiting period of a few hours is allowed to elapse, during which time aminolevulinic acid will be taken up by cells, and aminolevulinic acid will be converted by the cells to protoprophyrin IX, a photosensitizer.

    [0089] The physician shines a bright red light (from an array of light-emitting diodes or a diode laser) on the area to be treated. The light exposure lasts a few minutes to a few tens of minutes. Protoprophyrin IX absorbs light, exciting it to an excited singlet state.

    [0090] Intersystem crossing occurs, resulting in excited triplet protoprophyrin IX. Energy is transferred from triplet protoporphyrin IX to triplet oxygen, resulting in singlet (ground state) protoporphyrin IX and excited singlet oxygen. Singlet oxygen reacts with biomolecules, fatally damaging some cells in the treatment area. Within a few days, the exposed skin and carcinoma will scab over and flake away. In a few weeks, the treated area has healed, leaving healthy skin behind. For extensive malignancies, repeat treatments may be required. It is also common to experience pain from the area treated. After the treatment the patient will need to avoid excessive exposure to sunlight for a period of time.

    Example 2. Toxicity of ACT4211L

    [0091] LC50 determination was carried out as described in Lewis, Thomas J. Toxicity and cytopathogenic properties toward human melanoma cells of activated cancer therapeutics in zebra fish. Integrative Cancer Therapies 9.1 (2010): 84-92. Briefly, 20 hpf zebrafish (n=30) were treated with ACT4211L at: 100, 200, 300, 400, 500, 600, 750, 850, 1000, 1500 and 2000 M for 28 hours at 28 C. and lethality was recorded at 48 hpf. Significant lethality was not observed (FIG. 1). No significant lethality was observed in the repeat experiment. No further testing was performed.

    [0092] Assessment of cytotoxicity for melanoma cancer cell line WM-266-4. ACT4211L exhibited significant cytotoxic effect on human melanoma cancer cells WM-266-4 in vitro. A dose response effect was observed (FIG. 2); 5.5, 42.7, 56.0, and 64.8% cell death was observed at: 1, 10, 100 and 1000 M concentration, respectively.

    TABLE-US-00011 TABLE I Results of LC50 determination. # # Concen- Dead Dead Mean tration Fish Mortality Fish Mortality Mortality Survival (M) (exp 1) (%) (exp 2) (%) (%) (%) 0 1 3.3 0 0.0 1.7 98.3 100 0 0.0 0 0.0 0.0 100.0 200 0 0.0 0 0.0 0.0 100.0 300 0 0.0 0 0.0 0.0 100.0 400 1 3.3 0 0.0 1.7 98.3 500 0 0.0 0 0.0 0.0 100.0 600 1 3.3 0 0.0 1.7 98.3 750 0 0.0 0 0.0 0.0 100.0 850 0 0.0 0 0.0 0.0 100.0 1000 0 0.0 0 0.0 0.0 100.0 1500 0 0.0 0 0.0 0.0 100.0 2000 3 10.0 0 0.0 5.0 95.0

    TABLE-US-00012 TABLE II One-way analysis of variance (One-way ANOVA) P value 0.0001 Are means signif. different? (P < 0.05) Yes Number of groups 6 Dunnett's Multiple Mean Comparison Test Diff. q P value 95% CI of diff 0 vs 0.1 7849 2.185 P > 0.05 1706 to 17400 0 vs 1 10570 2.942 P < 0.05 1015 to 20120 0 vs 10 12740 3.547 P < 0.01 3186 to 22300 0 vs 100 16680 4.644 P < 0.01 7126 to 26240 0 vs 1000 19300 5.373 P < 0.01 9745 to 28850

    TABLE-US-00013 TABLE III Results of cytotoxicity assessment for human melanoma cancer cell WM-266-4 % % of SD SE%/ ACT4211L of Cell % of of (M) Mean SD Control Death Control SE Control 0 29806 7194 100.0 0.0 24.1 2936 9.9 0.1 21958 9590 73.7 26.3 32.2 3914 1.1 1 19237 5279 64.5 35.5 17.7 2155 7.2 10 27066 7482 57.3 42.7 25.1 3054 10.2 100 13125 1938 44.0 56.0 6.5 791 2.7 1000 10506 956 35.2 64.8 3.2 390 1.3

    Example 3. Toxicity of Compound ACT4211

    [0093] LC50 determination was made as described in Lewis, Thomas J. Toxicity and cytopathogenic properties toward human melanoma cells of activated cancer therapeutics in zebra fish. Integrative Cancer Therapies 9.1 (2010): 84-92. Briefly, 20 hpf zebrafish (n=30) were treated with ACT4211 at: 1, 10, 100, 1000 and 2000 M for 28 hours at 28 C. and lethality was recorded at 48 hpf. No lethality was observed up to 2000 M (FIG. 3). No further testing was performed.

    [0094] Assessment of ACT4211 cytotoxicity for melanoma cancer cell line WM-266-4: ACT4211exhibited significant cytotoxic effect on human melanoma cancer cells WM-266-4 in vitro. A dose response effect was observed; 57%, 81%, and 87% cell death was observed at: 100, 1000 and 2000 M concentration, respectively (FIG. 4).

    TABLE-US-00014 TABLE IV Results of LC50 determination-ACT4211 # # Mean Concen- Dead Mor- Dead Mor- Mor- tration Fish tality Fish tality tality Survival (M) (exp 1) (%) (exp 2) (%) (%) (%) 0 0 3.3 0 0.0 0.0 100.0 1 0 0.0 0 0.0 0.0 100.0 10 0 0.0 0 0.0 0.0 100.0 100 0 0.0 0 0.0 0.0 100.0 1000 0 0.0 0 0.0 0.0 100.0 2000 0 0.0 0 0.0 0.0 100.0

    TABLE-US-00015 TABLE V One-way analysis of variance (One-way ANOVA) P value <0.0001 Are means signif. different? (P < 0.05) Yes Number of groups 6 Dunnett's Multiple Mean Comparison Test Diff. q P value 95% CI of diff 0 vs 1 1641 2.565 P > 0.05 60.52 to 3343 0 vs 10 69.17 0.1081 P > 0.05 1633 to 1771 0 vs 100 2330 3.642 P < 0.01 628.0 to 4032 0 vs 1000 4119 6.438 P < 0.01 2417 to 5821 0 vs 2000 4631 7.238 P < 0.01 2929 to 6333

    TABLE-US-00016 TABLE VI Results of cytotoxicity assessment for human melanoma cancer cell WM-266-4 ACT4211 Mean SD % of Conc. (uM) (cell #) (Cell #) cell death SD(%) SE (%) 0 344034 79063 0 23 9.4 1 359326 111434 4 32 13.2 10 335549 78258 2 23 9.3 100 147444 24512 57 7 2.9 1000 66851 9354 81 3 1.1 2000 43788 10132 87 3 1.2

    TABLE-US-00017 TABLE VI Results of cytotoxicity assessment for human melanoma cancer cell WM-266-4 ACT4211 Mean SD % of Conc. (uM) (cell #) (Cell #) cell death SD(%) SE (%) 0 344034 79063 0 23 9.4 1 359326 111434 4 32 13.2 10 335549 78258 2 23 9.3 100 147444 24512 57 7 2.9 1000 66851 9354 81 3 1.1 2000 43788 10132 87 3 1.2

    Example 4. Studies of Compound ACT4211

    [0095] In this study the effect of SDT with ACT4211 on S-180 sarcoma in mice was examined, as described in Wang, Xiaohuai, Thomas J. Lewis, and Doug Mitchell. The tumoricidal effect of sonodynamic therapy (SDT) on S-180 sarcoma in mice. Integrative cancer therapies 7.2 (2008): 96-102. Tumor growth inhibition was visible even when covered by barrier of bone. Pathological slices showed coagulated necrosis or metamorphic tissue with inflammatory reaction in the tumor taken from 2 hours to 36 hours after SDT. These data revealed that SDT with ACT4211 inhibited growth of mouse S-180 sarcoma and the inhibitive effect was sound intensity dependent. SDT also induced some inflammation while it destroyed the tumor, indicative of a vaccine affect. ACT4211 shows great promise for clinical use in the future.

    TABLE-US-00018 TABLE 1 Tumor weight in each group 15 days after treatment Group Mean of tumor weight (g) P (Comparing with C) C 0.361 0.094 U 0.440 0.275 >0.05 S 0.272 0.328 >0.05 SU 0.009 0.003 <0.01

    [0096] Comparing with group C (control), the tumor weight in group SU was significantly lower (P<0.01). The tumor weight in group U and S had no significant difference like that of group C. This demonstrated that the ACT4211 plus sound treatment inhibited S-180 sarcoma in mice.

    TABLE-US-00019 TABLE 2 Tumor size in each group 15 days after treatment Group Mean of tumor size (cm.sup.3) P (Comparing with C) C 0.865 0.124 U 0.799 0.315 >0.05 S 0.611 0.190 >0.05 SU 0.047 0.019 <0.01

    [0097] As Table 3 shows, the tumor weight in the three SDT treated groups was much lower than that in Control group (P<0.05). These results conform to the conclusion that SDT with ACT4211 inhibits S-180 sarcoma in mice. It is very clear that the higher intensity of ultrasound used, the higher inhibitive response was produced at the range of ultrasound intensity from 0.3 W/cm2 to 1.2 W/cm2.

    TABLE-US-00020 TABLE 3 Tumor weight in each group 15 days after treatment Mean of tumor P (Comparing P (Comparing Group weight (g) with Control) with SU1) Control 0.361 0.094 <0.05 SU1 0.0425 0.025 <0.05 SU2 0.021 0.006 <0.01 <0.05 SU3 0.009 0.003 <0.01 <0.01

    [0098] Tumor size was measured with sliding calipers every one or two days. The results are shown in Table 4.

    TABLE-US-00021 TABLE 4 the tumor size in each group 15 days after treatment Mean of tumor P(Comparing Group size (cm.sup.3) with SU1) Control 0.865 0.124 <0.05 SU1 0.383 0.113 SU2 0.118 0.020 <0.05 SU3 0.047 0.019 <0.01

    [0099] As Table 4 shows, the tumors in the 3 SDT treatment groups were much smaller than that in Control group (P<0.05). The inhibitive effect of SDT with ACT4211 was sound intensity dependent.

    [0100] The pathological study results in group SU (ACT4211 20 mg/Kg and ultrasound of 1.2 W/cm2) also showed superior results. Pathological slices were made from the mice sacrificed at 2 hours, 36 hours and 15 days after SDT. Coagulated necrosis or metamorphic tissue with inflammatory reaction in the tumor was observed and that the processes of necroses, degeneration and inflammation were further enhanced 36 hours after the SDT treatment. Fifteen days after SDT, only coagulated necroses and vacuole degeneration was visible in the tumor, but no living tumor cells could be identified. There was some inflammation and fibrosis around the necrotic or degenerative tumor. These data revealed that SDT with ACT4211 destroyed the S-180 sarcoma mouse very rapidly. The degeneration of tumor induced by SDT occurred almost immediately or at least within two hours after SDT treatment. These data also revealed that along with the necroses and degeneration of the tumor, SDT also induced inflammatory reaction in the tumor and the reaction may last for seven days. Observations with confocal laser scanning microscopy suggest that ACT4211 accumulates specifically within tumor cells.

    [0101] SDT with a piece of bone between tumor and ultrasound was performed as described in Wang, Xiaohuai, Thomas J. Lewis, and Doug Mitchell. The tumoricidal effect of sonodynamic therapy (SDT) on S-180 sarcoma in mice. Integrative cancer therapies 7.2 (2008): 96-102. The results are shown in Table 5.

    TABLE-US-00022 TABLE 5 Tumor weight in each group 15 days after treatment Mean of tumor P(Comparing Group weight (g) with C) C 0.73466 0.0781 SU 0.07416 0.0158 >0.01

    [0102] SDT with a piece of bone between tumor and ultrasound was still able to inhibit the tumor growth. This revealed that 1 MHz ultrasound can pass through bone, activate the sensitizer in the tumor and lead to tumor destruction.

    [0103] The inhibitive effect of SDT with different ultrasound frequency on S-180 sarcoma in mice Was carried out as described in Wang, Xiaohuai, Thomas J. Lewis, and Doug Mitchell. The tumoricidal effect of sonodynamic therapy (SDT) on S-180 sarcoma in mice. Integrative cancer therapies 7.2 (2008): 96-102. The results are shown in Table 6.

    TABLE-US-00023 TABLE 6 Tumor weight in each group 8 days after treatment Mean of tumor P (Comparing P(Comparing Group weight (g) with C) with SU2) C 0.43208 0.128413 SU1 0.12515 0.019856 <0.01 >0.05 SU2 0.111967 0.031018 <0.01 SU3 0.121633 0.020449 <0.01 >0.05

    [0104] Compared to group C, the tumor weight in every SDT treated groups was significantly lower (P<0.01). This demonstrated again that the ACT4211 plus sound treatment did inhibit S-180 sarcoma in mice. The data suggests that 0.5 to 2.5 MHz ultrasounds were all able to active ACT4211 and destroy the tumor.

    Example 5. Sono-Photodynamic Therapy

    [0105] A dose of 45 mg of photo/sonosensitizer is administered sublingually over 2 to 5 hours. No photosensitivity from normal ambient, artificial, or natural light has been noted but as a precaution patients arc advised not to stay in direct sunlight for periods over half an hour for one week following sensitizer administration. After 48 hours the patient is then exposed to a light bed containing 48 panels, each with 1028 LED's emitting a combination of visible and infra-red light at the frequencies 635 nm and 820 nm. Light bed exposure varies from two sessions of 2 to 15 minutes per day with shorter exposure duration in cases with larger tumor load. Ultrasound is applied using a single maniple at 1 W/cm2 and a frequency of 1 MHz at sites of known malignant disease for 10-30 minutes total. Light and ultrasound activation is repeated on three consecutive days. Ozone auto-haemotherapy (40 IU) is administered immediately before light bed exposure. Further the sensitizer is usually administered after one week for a second treatment cycle. Dexamethasone is administered to some patients with significant tumor load, with dosage titrated on a case-by-case basis.

    [0106] Other examples are found in Kenyon, Julian N., Richard James Fuller, and Thomas Joseph Lewis. Activated cancer therapy using light and ultrasound-A case series of sonodynamic photodynamic therapy in 115 patients over a 4 year period. Current Drug Therapy 4.3 (2009): 179-193 and Wang, Xiaohuai, et al. Sonodynamic and photodynamic therapy in advanced breast carcinoma: a report of 3 cases. Integrative Cancer Therapies 8.3 (2009): 283-287.

    [0107] While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims.