U.S. patent application number 10/315319 was filed with the patent office on 2003-10-16 for therapeutic agents - iii.
Invention is credited to Aylward, James Harrison, Parsons, Peter Gordon, Suhrbier, Andreas, Turner, Kathleen Anne.
Application Number | 20030195168 10/315319 |
Document ID | / |
Family ID | 3822089 |
Filed Date | 2003-10-16 |
United States Patent
Application |
20030195168 |
Kind Code |
A1 |
Aylward, James Harrison ; et
al. |
October 16, 2003 |
Therapeutic agents - III
Abstract
The present invention relates generally to chemical agents
useful in the treatment and prophylaxis of protein kinase C (PKC)
related conditions in mammals, including humans and primates,
non-mammalian animals and avian species. More particularly, the
present invention provides a chemical agent of the macrocyclic
diterpene family obtainable from a member of the Euphorbiaceae
family of plants or botanical or horticultural relatives thereof or
derivatives or chermical analogues or chemically synthetic forms of
the agents for use in the treatment or prophylaxis of PKC-related
conditions in mammalian, animal and avian subjects. The subject
chemical agents are also useful for modulating expression of
genetic sequences including promotion and other regulatory
sequences. The present invention further contemplates a method for
the prophylaxis and/or treatment in mammalian, animal or avian
subjects with PKC-related conditions by the topical or systemic
administration of a macrocyclic diterpene obtainable from a member
of the Euphorbiaceac family of plants or their botanical or
horticultural derivatives or a derivative, chemical analogue or
chemically synthetic form of the agent. The chemical agent of the
present invention may be in the form of a purified compound,
mixture of compounds, a precursor form of one or more of the
compounds capable of chemical transformation into a therapeutically
and/or genetically active agent or in the form of a chemical
fraction, sub-fraction, preparation or extract of the plant.
Inventors: |
Aylward, James Harrison;
(Queensland, AU) ; Parsons, Peter Gordon;
(Queensland, AU) ; Suhrbier, Andreas; (Queensland,
AU) ; Turner, Kathleen Anne; (Victoria, AU) |
Correspondence
Address: |
Scully, Scott, Murphy & Presser
400 Garden City Plaza
Garden City
NY
11530
US
|
Family ID: |
3822089 |
Appl. No.: |
10/315319 |
Filed: |
December 9, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10315319 |
Dec 9, 2002 |
|
|
|
PCT/AU01/00678 |
Jun 7, 2001 |
|
|
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Current U.S.
Class: |
514/63 ; 514/114;
514/430; 514/475; 514/549; 514/64 |
Current CPC
Class: |
A61P 37/04 20180101;
A61P 39/02 20180101; Y02A 50/483 20180101; Y02A 50/491 20180101;
A61P 11/06 20180101; A61P 31/18 20180101; A61P 29/00 20180101; A61P
31/00 20180101; A61K 31/455 20130101; A61P 7/00 20180101; Y02A
50/473 20180101; Y02A 50/478 20180101; Y02A 50/30 20180101; A61P
19/02 20180101; A61P 35/00 20180101; Y02A 50/469 20180101; A61P
3/10 20180101; A61P 17/06 20180101; Y02A 50/471 20180101; A61P
31/02 20180101; Y02A 90/26 20180101; A61K 36/47 20130101; A61P
31/04 20180101; A61P 9/00 20180101; Y02A 50/414 20180101; A61P
17/00 20180101; A61P 25/28 20180101; A61P 31/14 20180101; A61P
37/08 20180101; A61P 31/16 20180101; A61P 31/22 20180101; Y02A
50/415 20180101; Y02A 90/10 20180101; C07C 69/533 20130101; A61P
17/10 20180101; Y02A 50/409 20180101; A61P 19/00 20180101; A61P
25/00 20180101; Y02A 50/463 20180101; A61P 37/06 20180101; A61P
43/00 20180101; Y02A 50/465 20180101; Y02A 50/475 20180101; A61P
17/04 20180101; A61P 39/00 20180101; Y02A 50/401 20180101; A61P
9/12 20180101; A61K 31/22 20130101; A61P 31/12 20180101; A61P 31/20
20180101; A61P 33/00 20180101; A61P 9/10 20180101; A61P 25/24
20180101; A61P 33/10 20180101; Y02A 50/402 20180101 |
Class at
Publication: |
514/63 ; 514/64;
514/549; 514/475; 514/430; 514/114 |
International
Class: |
A61K 031/695; A61K
031/69; A61K 031/66; A61K 031/22; A61K 031/38 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 7, 2000 |
AU |
PQ8017 |
Claims
1. A method for the treatment or prophylaxis of a PKC-related
condition or disorder in a subject, said method comprising the
administration to said subject of a symptom-ameliorating effective
amount of a chemical agent obtainable from a plant of the
Euphorbiaceae family or a derivative or chemical analogue thereof
which chemical agent is a macrocyclic diterpene selected from
compounds of the ingenane, pepluane and jatrophane families and
which chemical agent or derivative or chemical analogue is
represented by any one of the general formulae (I)-(V): 7wherein: n
is 0-10 atoms selected from carbon, oxygen, nitrogen, sulfur,
phosphorus, silicon, boron, arsenic and selenium, wherein the ring
defined by said atoms is saturated or unsaturated, including
epoxides and thioepoxides; A-T are independently selected from
hydrogen, R.sub.1, R.sub.2, R.sub.3, F, Cl, Br, I, CN, OR.sub.1,
SR.sub.1, NR.sub.1R.sub.2, N(.dbd.O).sub.2, NR.sub.1OR.sub.2,
ONR.sub.1R.sub.2, SOR.sub.1, SO.sub.2R.sub.1, SO.sub.3R.sub.1,
SONR.sub.1R.sub.2, SO.sub.2NR.sub.1R.sub.2,
SO.sub.3NR.sub.1R.sub.2, P(R.sub.1).sub.3,
P(.dbd.O)(R.sub.1).sub.3, Si(R.sub.1).sub.3, B(R.sub.1).sub.2,
(C.dbd.X)R.sub.3 or X(C.dbd.X)R.sub.3 where X is selected from
sulfur, oxygen and nitrogen; R.sub.1 and R.sub.2 are each
independently selected from C.sub.1-C.sub.20 alkyl (branched and/or
straight chained), C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and/or straight chained), C.sub.2-C.sub.10 alkynyl (branched and/or
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.1, SR.sub.1, NR.sub.1R.sub.2, N(.dbd.O).sub.2,
NR.sub.1OR.sub.2, ONR.sub.1R.sub.2, SOR.sub.1, SO.sub.2R.sub.1,
SO.sub.3R.sub.1, SONR.sub.1R.sub.2, SO.sub.2NR.sub.1R.sub.2,
SO.sub.3NR.sub.1R.sub.2, P(R.sub.1).sub.3,
P(.dbd.O)(R.sub.1).sub.3, Si(R.sub.1).sub.3,
B(R.sub.1).sub.2]alkyl; R.sub.3 is selected from R.sub.1, R.sub.2,
CN, COR.sub.1, CO.sub.2R.sub.1, OR.sub.1, SR.sub.1,
NR.sub.1R.sub.2, N(.dbd.O).sub.2, NR.sub.1OR.sub.2,
ONR.sub.1R.sub.2, SOR.sub.1, SO.sub.2R.sub.1, SO.sub.3R.sub.1,
SONR.sub.1R.sub.2, SO.sub.2NR.sub.1R.sub.2,
SO.sub.3NR.sub.1R.sub.2, P(R.sub.1).sub.3,
P(.dbd.O)(R.sub.1).sub.3, Si(R.sub.1).sub.3, B(R.sub.1).sub.2; A
connected to B (or C), D (or E, R (or Q), P (or O) or S (or T) is a
selection of C.sub.1-C.sub.8 disubstituted (fused) saturated or
unsaturated carbocyclic or heterocyclic rings further substituted
by R.sub.3, (C.dbd.X)R.sub.3 and X(C.dbd.X)R.sub.3, including
epoxides and thioepoxides; J connected to I (or H), G (or F), K (or
L), M (or N) or S (or T) is a selection of C.sub.1-C.sub.8
disubstituted (fused) saturated and unsaturated carbocyclic or
heterocyclic rings further substituted by R.sub.3, (C.dbd.X)R.sub.3
and X(C.dbd.X)R.sub.3, including epoxides and thioepoxides; D (or
E) connected to B (or C) or G (or F); I (or H) connected to G (or
F); P (or O) connected to R (or Q) or M (or N); K (or L) connected
to N (or M) is a selection of C.sub.1-C.sub.8 disubstituted (fused)
saturated or unsaturated carbocyclic or heterocyclic rings
substituted by R.sub.3, (C.dbd.X)R.sub.3 and X(C.dbd.X)R.sub.3,
including epoxides and thioepoxides; B and C, D and E, R and Q, P
and O, I and H, G and F, K and L, M and N or S and T are .dbd.X
where X is selected from sulfur, oxygen, nitrogen, NR.sub.1R.sub.2,
and .dbd.CR.sub.1R.sub.2 8wherein: n is 0-10 atoms selected from
carbon, oxygen, nitrogen, sulfur, phosphorus, silicon, boron,
arsenic and selenium, wherein the ring defined by said atoms is
saturated or unsaturated, including epoxides and thioepoxides;
A'-T' are independently selected from hydrogen, R.sub.4, R.sub.5,
R.sub.6, F, Cl, Br, I, CN, COR.sub.4, CO.sub.2R.sub.4, OR.sub.4,
SR.sub.4, NR.sub.4R.sub.5, CONR.sub.4R.sub.5, N(.dbd.O).sub.2,
NR.sub.4OR.sub.5, ONR.sub.4R.sub.5, SOR.sub.4, SO.sub.2R.sub.4,
SO.sub.3R.sub.4, SONR.sub.4R.sub.5, S0.sub.2NR4R.sub.5,
S0.sub.3NR.sub.4R.sub.5, P(R.sub.4).sub.3,
P(.dbd.O)(R.sub.4).sub.3, Si(R.sub.4).sub.3, B(R.sub.4).sub.2,
(C.dbd.X)R.sub.6 or X(C.dbd.X)R.sub.6 where X is selected from
sulfur, oxygen and nitrogen; R.sub.4 and R.sub.5 are each
independently selected from C.sub.1-C.sub.20 alkyl (branched and/or
straight chained), C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and/or straight chained), C.sub.2-C.sub.10 alkynyl (branched and/or
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.4, SR.sub.4, NR.sub.4R.sub.5, N(.dbd.O).sub.2,
NR.sub.4OR.sub.5, ONR.sub.4R.sub.5, SOR.sub.4, SO.sub.2R.sub.4,
SO.sub.3R.sub.4, SONR.sub.4R.sub.5, SO.sub.2NR.sub.4R.sub.5,
SO.sub.3NR.sub.4R.sub.5, P(R.sub.4).sub.3,
P(.dbd.O)(R.sub.4).sub.3, Si(R.sub.4).sub.3,
B(P.sub.4).sub.2]alkyl; R.sub.6 is selected from R.sub.4, R.sub.5,
CN, COR.sub.4, CO.sub.2R.sub.4, OR.sub.4, SR.sub.4,
NR.sub.4R.sub.5, N(.dbd.O).sub.2, NR.sub.4OR.sub.5,
ONR.sub.4R.sub.5, SOR.sub.4, SO.sub.2R.sub.4, SO.sub.3R.sub.4,
SONR.sub.4R.sub.5, SO.sub.2NR.sub.4R.sub.5,
SO.sub.3NR.sub.4R.sub.5, P(R.sub.4).sub.3,
P(.dbd.O)(R.sub.4).sub.3, Si(R.sub.4).sub.3, B(R.sub.4).sub.2; E'
and R' or H' and O' is a C.sub.2-C.sub.8 saturated or unsaturated
carbocyclic or heterocyclic ring system further substituted by
R.sub.6, including epoxides and thioepoxides; O' connected to M'
(or N') or Q' (or P'); R' connected to Q' (or P') or S' (or T'); S'
(or T') connected to A' (or B'); A' (or B') connected to C' (or
D'); E' connected to C' (or D') or F' (or G'); H' connected to I';
I' connected to J'; J' connected to K'; K' connected to L'; L'
connected to M' (or N') are C.sub.1-C.sub.8 disubstituted (fused)
saturated or unsaturated carbocyclic or heterocyclic ring systems
further substituted by R.sub.6, (C.dbd.X)R.sub.6 and
X(C.dbd.X)R.sub.6, including epoxides and thioepoxides; A', B' and
C', D' and F', G' and M', N' and P', Q' and S', T' are .dbd.X where
X is selected from sulfur, oxygen, nitrogen, NR.sub.4R.sub.5,
(C.dbd.X)R.sub.6, X(C.dbd.X)R.sub.6, and .dbd.CR.sub.7R.sub.8;
R.sub.7 and R.sub.8 are each independently selected from R.sub.6,
(C.dbd.X)R.sub.6 and X(C.dbd.X)R.sub.6 9wherein: n is 0-10 atoms
selected from carbon, oxygen, nitrogen, sulfur, phosphorus,
silicon, boron, arsenic and selenium, wherein the ring defined by
said atoms is saturated or unsaturated, including epoxides and
thioepoxides; A.sup.1-T.sup.1 are independently selected from
hydrogen, R.sub.9, R.sub.10, R.sub.11, F, Cl, Br, I, CN, OR.sub.9,
SR.sub.9, NR.sub.9R.sub.10, N(.dbd.O).sub.2, NR.sub.9OR.sub.10,
ONR.sub.9R.sub.10, SOR.sub.9, SO.sub.2R.sub.9, SO.sub.3R.sub.9,
SONR.sub.9R.sub.10, SO.sub.2NR.sub.9R.sub.10,
SO.sub.3NR.sub.9R.sub.10, P(R.sub.9).sub.3,
P(.dbd.O)(R.sub.9).sub.3, Si(R.sub.9).sub.3, B(R.sub.9).sub.2,
(C.dbd.X)R.sub.11 or X(C.dbd.X)R.sub.11 where X is selected from
sulfur, oxygen and nitrogen; R.sub.9 and R.sub.10 are each
independently selected from C.sub.1-C.sub.20 alkyl (branched and
straight chained), C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and straight chained), C.sub.2-C.sub.10 alkynyl (branched and
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.9, SR.sub.9, NR.sub.9R.sub.10, N(.dbd.O).sub.2,
NR.sub.9OR.sub.10, ONR.sub.9R.sub.10, SOR.sub.9, SO.sub.2R.sub.9,
SO.sub.3R.sub.9, SONR.sub.9R.sub.10, SO.sub.2NR.sub.9R.sub.10,
SO.sub.3NR.sub.9R.sub.10, P(R.sub.9).sub.3,
P(.dbd.O)(R.sub.9).sub.3, Si(R.sub.9).sub.3,
B(R.sub.9).sub.2]alkyl; R.sub.11 is selected from R.sub.9,
R.sub.10, CN, COR.sub.9, CO.sub.2R.sub.9, OR.sub.9, SR.sub.9,
NR.sub.9R.sub.10, N(.dbd.O).sub.2, NR.sub.9OR.sub.10,
ONR.sub.9R.sub.10, SOR.sub.9, SO.sub.2R.sub.9, SO.sub.3R.sub.9,
SONR.sub.9R.sub.10, SO.sub.2NR.sub.9R.sub.10,
SO.sub.3NR.sub.9R.sub.10, P(R.sub.9).sub.3,
P(.dbd.O)(R.sub.9).sub.3, Si(R.sub.9).sub.3, B(R.sub.9).sub.2;
B.sup.1 and R.sup.1, E.sup.1 and .sup.1 and .sup.1 and M.sup.1 are
selected from a C.sub.2-C.sub.8 saturated or unsaturated
carbocyclic or heterocyclic ring system further substituted by
R.sub.11, including epoxides and thioepoxides; A.sup.1 (or .sup.1
connected to .sup.1 (or .sup.1) or T.sup.1 (or S.sup.1) B.sup.1
connected to .sup.1 (or .sup.1) or C.sup.1 (or D.sup.1) E.sup.1
connected to .sup.1 or C.sup.1 (or D.sup.1); .sup.1 connected to
.sup.1 (or F.sup.1); G.sup.1 (or H.sup.1) connected to .sup.1 (or
F.sup.1) or I.sup.1 (or J.sup.1); K.sup.1 (or L.sup.1) connected to
I.sup.1 (or J.sup.1) or M.sup.1; M.sup.1 connected to O.sup.1 (or
N.sup.1); .sup.1 connected O.sup.1 (or N.sup.1) or P.sup.1 (or
Q.sup.1); R.sup.1 connected P.sup.1 (or Q.sup.1) or S.sup.1 (or
T.sup.1) are C.sub.1-C.sub.8 disubstituted (fused) saturated or
unsaturated carbocyclic or heterocyclic ring systems further
substituted by R.sub.11, (C.dbd.X)R.sub.11 and X(C.dbd.X)R.sub.11,
including epoxides and thioepoxides; A.sup.1, and , and C.sup.1,
D.sup.1 and F.sup.1, and G.sup.1, H.sup.1 and I.sup.1, J.sup.1 and
K.sup.1, L.sup.1 and N.sup.1, O.sup.1 and P.sup.1, Q.sup.1 and
S.sup.1, T.sup.1 are .dbd.X where X is selected from sulfur,
oxygen, nitrogen, NR.sub.9R.sub.10, including (C.dbd.X)R.sub.11 and
X(C.dbd.X)R.sub.11, and .dbd.CR.sub.12R.sub.13; R.sub.12 and
R.sub.13 are independently selected from R.sub.11,
(C.dbd.X)R.sub.11 and X(C.dbd.X)R.sub.11 10wherein: n is 0-10 atoms
selected from carbon, oxygen, nitrogen, sulfur, phosphorus,
silicon, boron, arsenic and selenium, wherein the ring defined by
said atoms is saturated or unsaturated, including epoxides and
thioepoxides; A.sup.2-X.sup.2 are independently selected from
hydrogen, R.sub.14, R.sub.15, R.sub.16, F, Cl, Br, I, CN,
OR.sub.14, SR.sub.14, NR.sub.14R.sub.15, N(.dbd.O).sub.2,
NR.sub.14OR.sub.15, ONR.sub.14R.sub.15, SOR.sub.14,
SO.sub.2R.sub.14, SO.sub.3R.sub.14, SONR.sub.14R.sub.15,
SO.sub.2NR.sub.14R.sub.15, SO.sub.3NR.sub.14R.sub.15- ,
P(R.sub.14).sub.3, P(.dbd.O)(R.sub.14).sub.3, Si(R.sub.14).sub.3,
B(R.sub.14), (C.dbd.Y)R.sub.16 or Y(C.dbd.Y)R.sub.16 where Y is
selected from sulfur, oxygen and nitrogen; R.sub.14 and R.sub.15
are each independently selected from C.sub.1-C.sub.20 alkyl
(branched and/or straight chained), C.sub.1-C.sub.20 arylalkyl,
C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14
heteroaryl, C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl
(branched and/or straight chained), C.sub.2-C.sub.10 alkynyl
(branched and/or straight chained), C.sub.1-C.sub.10
heteroarylalkyl, C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10
haloalkyl, dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10
[CN, OR.sub.14, SR.sub.14, NR.sub.14R.sub.10, N(.dbd.O).sub.2,
NR.sub.14OR.sub.15, ONR.sub.14R.sub.15, SOR.sub.14,
SO.sub.2R.sub.14, SO.sub.3R.sub.14, SONR.sub.14R.sub.15,
SO.sub.2NR.sub.14R.sub.15, SO.sub.3NR.sub.14R.sub.15,
P(R.sub.14).sub.3, P(.dbd.O)(R.sub.14).sub.3, Si(R.sub.14).sub.3,
B(R.sub.14).sub.2]alkyl; R.sub.16 is selected from R.sub.14,
R.sub.15, CN, COR.sub.14, CO.sub.2R.sub.15, OR.sub.14, SR.sub.14,
NR.sub.14R.sub.15, N(.dbd.O).sub.2, NR.sub.14OR.sub.15,
ONR.sub.14R.sub.15, SOR.sub.14, SO.sub.2R.sub.14, SO.sub.3R.sub.14,
SONR.sub.14R.sub.15, SO.sub.2NR.sub.14R.sub.15,
SO.sub.3NR.sub.14R.sub.15, P(R.sub.14).sub.3,
P(.dbd.O)(R.sub.14).sub.3, Si(R.sub.14).sub.3, B(R.sub.14).sub.2;
E.sup.2 and V.sup.2, H.sup.2 and S.sup.2, and I.sup.2 and P.sup.2
are C.sub.2-C.sub.8 saturated or unsaturated carbocyclic or
heterocyclic ring system further substituted by R.sub.16, including
epoxides and thioepoxides; A.sup.2 (or B.sup.2) connected to
C.sup.2 (or D.sup.2) or W.sup.2 (or X.sup.2); E.sup.2 connected to
C.sup.2 (or D.sup.2) or F.sup.2 (or G.sup.2); H.sup.2 connected to
F.sup.2 (or G.sup.2) or I.sup.2; I.sup.2 connected to J.sup.2 (or
K.sup.2); L.sup.2 (or M.sup.2) connected to J.sup.2 (or K.sup.2) or
N.sup.2 (or O.sup.2); R.sup.2 (or Q.sup.2) connected to P.sup.2 or
S.sup.2; V.sup.2 connected to U.sup.2 (or T.sup.2) or W.sup.2 (or
X.sup.2) are C.sub.1-C.sub.8 disubstituted (fused) saturated or
unsaturated carbocyclic or heterocyclic ring systems further
substituted by R.sub.16, (C.dbd.Y)R.sub.16 and Y(C.dbd.Y)R.sub.16,
including epoxides and thioepoxides; A.sup.2, B.sup.2; C.sup.2,
D.sup.2; F.sup.2, G.sup.2; J.sup.2, K.sup.2; L.sup.2, M.sup.2;
N.sup.2, O.sup.2; Q.sup.2, R.sup.2; U.sup.2, T.sup.2 and X.sup.2,
W.sup.2 are .dbd.Y where Y is selected from sulfur, oxygen,
nitrogen, NR.sub.14R.sub.15 and .dbd.CR.sub.17R.sub.18; R.sub.17
and R.sub.18 are independently selected from R.sub.16,
(C.dbd.Y)R.sub.16 and Y(C.dbd.Y)R.sub.16 11wherein: n is 0-10 atoms
selected from carbon, oxygen, nitrogen, sulfur, phosphorus,
silicon, boron, arsenic and selenium, wherein the ring defined by
said atoms is saturated or unsaturated, including epoxides and
thioepoxides; A.sup.3-Z.sup.3 are independently selected from
hydrogen, R.sub.19, R.sub.20, R.sub.21, F, Cl, Br, I, CN,
OR.sub.19, SR.sub.19, NR.sub.19R.sub.20, N(.dbd.O).sub.2,
NR.sub.19OR.sub.20, ONR.sub.19R.sub.20, SOR.sub.19,
SO.sub.2R.sub.19, SO.sub.3R.sub.19, SONR.sub.19R.sub.20,
SO.sub.2NR.sub.19R.sub.20, SO.sub.3NR.sub.19R.sub.20,
P(R,.sub.9).sub.3, P(O)(R.sub.19).sub.3, Si(R.sub.19).sub.3,
B(R.sub.19).sub.2, (C.dbd..O slashed.)R.sub.21 or .O
slashed.(C.dbd..O slashed.)R.sub.21 where .O slashed. is sullur,
oxygen and nitrogen; R.sub.19 and R.sub.20 are each independently
selected from C.sub.1-C.sub.20 alkyl (branched and/or straight
chained), C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8 cycloalkyl,
C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and/or straight chained), C.sub.2-C.sub.10 alkynyl (branched and/or
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.19, SR.sub.19, NR.sub.19R.sub.20, N(.dbd.O).sub.2,
NR.sub.19OR.sub.20, ONR.sub.19R.sub.20, SOR.sub.19,
SO.sub.2R.sub.19, SO.sub.3R.sub.19, SONR.sub.19R.sub.20,
SO.sub.2NR.sub.19R.sub.20, SO.sub.3NR.sub.19R.sub.20,
P(R.sub.19).sub.3, P(.dbd.O)(R.sub.19).sub.3, Si(R.sub.19).sub.3,
B(R.sub.19).sub.2]alkyl; R.sub.21 is selected from R.sub.19,
R.sub.20, CN, COR.sub.19, CO.sub.2R.sub.19, OR.sub.19, SR.sub.19,
NR.sub.19R.sub.20, N(.dbd.O).sub.2, NR.sub.19OR.sub.20,
ONR.sub.19R.sub.20, SOR.sub.19, SO.sub.2R.sub.19, SO.sub.3R.sub.19,
SONR.sub.19R.sub.20, SO.sub.2NR.sub.19R.sub.20,
SO.sub.3NR.sub.19R.sub.20, P(R.sub.19).sub.3,
P(.dbd.O)(R.sub.19).sub.3, Si(R.sub.19).sub.3, B(R.sub.19).sub.2;
D.sup.3 connected to X.sup.3 is a C.sub.2-C.sub.8 saturated or
unsaturated carbocyclic or heterocyclic ring system further
substituted by R.sub.21, including epoxides and thioepoxides;
A.sup.3 (or .sup.3) connected to B.sup.3 (or C.sup.3) or E.sup.3
(or X); D.sup.3 connected to B.sup.3 (or C.sup.3) or E.sup.3 (or
F.sup.3); G.sup.3 (or H.sup.3) connected to E.sup.3 (or F.sup.3) or
I.sup.3 (or J.sup.3); L.sup.3 (or K.sup.3) connected to I.sup.3 (or
J.sup.3) or M.sup.3 (or N.sup.3); O.sup.3 (or .sup.3) connected to
N.sup.3 (or M.sup.3) or P.sup.3 (or Q.sup.3). S.sup.3 (or R.sup.3)
connected to Q.sup.3 (or P.sup.3) or U.sup.3 (or T.sup.3). W.sup.3
(or V.sup.3) connected to U.sup.3 (or T.sup.3) or X.sup.3; X.sup.3
connected to Y.sup.3 (or Z.sup.3) are C.sub.1-C.sub.8 disubstituted
(fused) saturated or unsaturated carbocyclic or heterocyclic ring
systems further substituted by R.sub.21, (C.dbd..O
slashed.)R.sub.21 and .O slashed. (C.dbd..O slashed.)R.sub.21,
including epoxides and thioepoxides; A.sup.3, .sup.3; B.sup.3,
C.sup.3; E.sup.3, F.sup.3; G.sup.3, H.sup.3; I.sup.3, J.sup.3;
K.sup.3, L.sup.3; M.sup.3, N.sup.3; O.sup.3, .sup.3; Q.sup.3,
P.sup.3, S.sup.3, R.sup.3, U.sup.3, T.sup.3, W.sup.3, V.sup.3, and
Z.sup.3, Y.sup.3 are .dbd..O slashed. where .O slashed. is selected
from sulfur, oxygen, nitrogen, NR.sub.19R.sub.20, and
.dbd.CR.sub.22R.sub.23; and R.sub.22 and R.sub.23 are selected from
R.sub.21, (C.dbd..O slashed.)R.sub.21 and .O slashed.(C.dbd..O
slashed.)R.sub.21; and which chemical agent or derivative or
chemical analogue thereof is capable of modulating PKC activity,
PKC-gene expression or PKC enzyme turnover and wherein said
chemical agent or its derivatives or chemical analogues is
administered for a time and under conditions sufficient to
ameliorate one or more symptoms associated with a PKC-related
condition or disorder.
2. A method according to claim 1 wherein the chemical agent is
represented by the general formula (VI): 12wherein: R.sub.24,
R.sub.25 and R.sub.26 are independently selected from hydrogen,
R.sub.27, R.sub.28, F, Cl, Br, I, CN, OR.sub.27, SR.sub.27,
NR.sub.27R.sub.28, N(.dbd.O).sub.2, NR.sub.27OR.sub.28,
ONR.sub.27R.sub.28, SOR.sub.27, SO.sub.2R.sub.27, SO.sub.3R.sub.27,
SONR.sub.27R.sub.28, SO.sub.2NR.sub.27R.sub.28,
SO.sub.3NR.sub.27R.sub.28, P(R.sub.27).sub.3,
P(.dbd.O)(R.sub.27).sub.3, Si(R.sub.27).sub.3, B(R.sub.27).sub.2,
(C.dbd.X)R.sub.29 or X(C.dbd.X)R.sub.29 where X is selected from
sulfur, oxygen and nitrogen; R.sub.27 and R.sub.28 are each
independently selected from C.sub.1-C.sub.20 alkyl (branched and/or
straight chained), C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and/or straight chained), C.sub.2-C.sub.10 alkynyl (branched and/or
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.27, SR.sub.27, NR.sub.27R.sub.28, N(.dbd.O).sub.2,
NR.sub.27OR.sub.28, ONR.sub.27R.sub.28, SOR.sub.27,
SO.sub.2R.sub.27, SO.sub.3R.sub.27, SONR.sub.27R.sub.28,
SO.sub.2NR.sub.27R.sub.28, SO.sub.3NR.sub.27R.sub.28,
P(R.sub.27).sub.3, P(.dbd.O)(R.sub.27).sub.3, Si(R.sub.27).sub.3,
B(R.sub.27).sub.2]alkyl; R.sub.29 is selected from R.sub.27,
R.sub.28, CN, COR.sub.27, CO.sub.2R.sub.27, OR.sub.27, SR.sub.27,
NR.sub.27R.sub.28, N(.dbd.O).sub.2, NR.sub.27OR.sub.28,
ONR.sub.27R.sub.28, SOR.sub.27, S0.sub.2R.sub.27, SO.sub.3R.sub.27,
SONR.sub.27R.sub.28, SO.sub.2NR.sub.27R.sub.28,
SO.sub.3NR.sub.27R.sub.28, P(R.sub.27).sub.3,
P(.dbd.O)(R.sub.27).sub.3, Si(R.sub.27).sub.3,
B(R.sub.27).sub.2.
3. A method according to claim 2 wherein R.sub.24 is H.
4. A method according to claim 2 wherein R.sub.24 is OAcetyl.
5. A method according to claim 2 wherein R.sub.24 is OH.
6. A method according to claim 2 wherein R.sub.25 and R.sub.26 are
OH.
7. A method according to claim 1 wherein the PKC-related condition
is alcoholism.
8. A method according to claim 1 wherein the PKC-related condition
is Alzheimer's disease.
9. A method according to claim 1 wherein the PKC-related condition
is asthma.
10. A method according to claim 1 wherein the PKC-related condition
is atherosclosis.
11. A method according to claim 1 wherein the PKC-related condition
is atopic dermatitis.
12. A method according to claim 1 wherein the PKC-related condition
is autoimmune disease.
13. A method according to claim 1 wherein the PKC-related condition
is bipolar disorder.
14. A method according to claim I wherein the PKC-related condition
is blood disorder.
15. A method according to claim 1 wherein the PKC-related condition
is cardiac hypertrophy.
16. A method according to claim 1 wherein the PKC-related condition
is depression.
17. A method according to claim 1 wherein the PKC-related condition
is diabetes.
18. A method according to claim 1 wherein the PKC-related condition
is hypertension.
19. A method according to claim 1 wherein the PKC-related condition
is hyperplastic dermatosis.
20. A method according to claim 1 wherein the PKC-related condition
is multiple sclerosis.
21. A method according to claim 1 wherein the PKC-related condition
is mycardial ischemia.
22. A method according to claim 1 wherein the PKC-related condition
is osteoarthritis.
23. A method according to claim 1 wherein the PKC-related condition
is psoriasis.
24. A method according to claim 1 wherein the PKC-related condition
is rheumatoid arthritis.
25. A method according to claim 1 wherein the PKC-related condition
is transplantation.
26. A method according to claim 1 wherein the PKC-related condition
is a latent virus.
27. A method according to claim 1 wherein the chemical agent is a
jatrophane or a derivative thereof or a pharmaceutically acceptable
salt of these.
28. A method according to claim 27 wherein said derivative is an
ester derivative.
29. A method according to claim 27 wherein said derivative is an
acetylated derivative.
30. A method according to claim 1 wherein said chemical agent is a
pepluane or a derivative thereof or a pharmaceutically acceptable
salt of these.
31. A method according to claim 30 wherein said derivative is an
ester derivative.
32. A method according to claim 30 wherein said derivative is an
acetylated derivative.
33. A method according to claim 1 wherein said chemical agent is a
paraliane or a derivative thereof or a pharmaceutically acceptable
salt of these.
34. A method according to claim 33 wherein said derivative is an
ester derivative.
35. A method according to claim 33 wherein said derivative is an
acetylated derivative.
36. A method according to claim 1 wherein said compound is an
angeloyl-substituted ingenane or a derivative thereof or a
pharmaceutically acceptable salt of these.
37. A method according to claim 36 wherein said derivative is an
acetylated derivative.
38. A method according to claim 36 wherein said jatrophane is of
conformation 2.
39. A method according to claim 27 or 30 or 33 or 36 wherein the
derivative comprises a substitution as represented in any one of
general formulae (I)-(VI).
40. A method according to claim 1 wherein said compound is
5,8,9,10,14-pentaacetoxy-3-benzoyloxy-15-hydroxypepluane (pepluane)
or a derivative thereof or a pharmaceutically acceptable salt of
these.
41. A method according to claim 40 wherein said derivative is an
ester derivative.
42. A method according to claim 1 wherein said compound is
2,3,5,7,15-pentaacetoxy-9-nicotinoyloxy-14-oxojatropha-6(17),11E-diene
(jatrophane 1) or a derivative thereof or a pharmaceutically
acceptable salt of these.
43. A method according to claim 42 wherein said derivative is an
ester derivative.
44. A method according to claim 1 wherein said compound is
2,5,7,8,9,14-hexaacetoxy-3-benzoyloxy-15-hydroxy-jatropha-6(17),11E-diene
(jatrophane 2) or a derivative thereof or a pharmaceutically
acceptable salt of these.
45. A method according to claim 44 wherein said derivative is an
ester derivative.
46. A method according to claim 1 wherein said compound is
2,5,14-triacetoxy-3-benzoyloxy-8,15-dihydroxy-7-isobutyroyloxy-9-nicotino-
yloxyjatropha-6(17), 11E-diene (jatrophane 3) or a derivative
thereof or a pharmaceutically acceptable salt of these.
47. A method according to claim 46 wherein said derivative is an
ester derivative.
48. A method according to claim 1 wherein said compound is
2,5,9,14-tetraacetoxy-3-benzoyloxy-8,15-dihydroxy-7-isobutyroyloxyjatroph-
a-6(17), 11E-diene) (jatrophane 4) or a derivative thereof or a
pharmaceutically acceptable salt of these.
49. A method according to claim 48 wherein said derivative is an
ester derivative.
50. A method according to claim 1 wherein said compound is
2,5,7,14-tetraacetoxy-3-benzoyloxy-8,15-dihydroxy-9-nicotinoyloxyjatropha-
-6(17),11E-diene (jatrophane 5) or a derivative thereof or a
pharmaceutically acceptable salt of these.
51. A method according to claim 50 wherein said derivative is an
ester derivative.
52. A method according to claim 1 wherein said compound is
2,5,7,9,14-pentaacetoxy-3-benzoyloxy-8,15-dihydroxyjatropha-6(17),
11E-diene (jatrophane 6) or a derivative thereof or a
pharmaceutically acceptable salt of these.
53. A method according to claim 52 wherein said derivative is an
ester derivative.
54. A method according to claim 1 wherein said compound is
20-O-acetyl-ingenol-3-angelate or a derivative thereof or a
pharmaceutically acceptable salt of these.
55. A method according to claim 54 wherein said derivative is an
ester derivative.
56. A method according to claim 27 or 30 or 33 or 36 or 40 or 42 or
44 or 46 or 48 or 50 or 52 or 54 wherein said compound is provided
in the form of a composition comprising a pharmaceutically- or
cosmetically-acceptable carrier.
57. A method according to claim 56 wherein said carrier is selected
from .beta.-alanine betaine hydrochloride and
t-4-hydroxy-N,N-dimethylproline.
58. A method of modulating the expression of a genetic sequence for
the treatment or prophylaxis of a condition or disorder in a
subject, said genetic sequence being under the control of a
promoter whose activity is modulated by a chemical agent obtainable
from a plant of the Euphorbiaceae family or a derivative or
chemical analogue thereof which chemical agent is a macrocyclic
diterpene selected from compounds of the ingenane, pepluane and
jatrophane families and which chemical agent or derivative or
chemical analogue is represented by any one of the general formulae
(I)-(V): 13wherein: n is 0-10 atoms selected from carbon, oxygen,
nitrogen, sulfur, phosphorus, silicon, boron, arsenic and selenium,
wherein the ring defined by said atoms is saturated or unsaturated,
including epoxides and thioepoxides; A-T are independently selected
from hydrogen, R.sub.1, R.sub.2, R.sub.3, F, Cl, Br, I, CN,
OR.sub.1, SR.sub.1, NR.sub.1R.sub.2, N(.dbd.O).sub.2,
NR.sub.1OR.sub.2, ONR.sub.1R.sub.2, SOR.sub.1, SO.sub.2R.sub.1,
SO.sub.3R.sub.1, SONR.sub.1R.sub.2, SO.sub.2NR.sub.1R.sub.2,
SO.sub.3NR.sub.1R.sub.2, P(R.sub.1).sub.3,
P(.dbd.O)(R.sub.1).sub.3, Si(R.sub.1).sub.3, B(R.sub.1).sub.2,
(C.dbd.X)R.sub.3 or X(C.dbd.X)R.sub.3 where X is selected from
sulfur, oxygen and nitrogen; R.sub.1 and R.sub.2 are each
independently selected from C.sub.1-C.sub.20 alkyl (branched and/or
straight chained), C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and/or straight chained), C.sub.2-C.sub.10 alkynyl (branched and/or
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.1, SR.sub.1, NR.sub.1R.sub.2, N(.dbd.O).sub.2,
NR.sub.1OR.sub.2, ONR.sub.1R.sub.2, SOR.sub.1, SO.sub.2R.sub.1,
SO.sub.3R.sub.1, SONR.sub.1R.sub.2, SO.sub.2NR.sub.1R.sub.2,
SO.sub.3NR.sub.1R.sub.2, P(R.sub.1).sub.3,
P(.dbd.O)(R.sub.1).sub.3, Si(R.sub.1).sub.3,
B(R.sub.1).sub.2]alkyl; R.sub.3 is selected from R.sub.1, R.sub.2,
CN, COR.sub.1, CO.sub.2R.sub.1, OR.sub.1, SR.sub.1,
NR.sub.1R.sub.2, N(.dbd.O).sub.2, NR.sub.1OR.sub.2,
ONR.sub.1R.sub.2, SOR.sub.1, SO.sub.2R.sub.1, SO.sub.3R.sub.1,
SONR.sub.1R.sub.2, SO.sub.2NR.sub.1R.sub.2,
SO.sub.3NR.sub.1R.sub.2, P(R.sub.1).sub.3,
P(.dbd.O)(R.sub.1).sub.3, Si(R.sub.1).sub.3, B(R.sub.1).sub.2; A
connected to B (or C), D (or E, R (or Q), P (or O) or S (or T) is a
selection of C.sub.1-C.sub.8 disubstituted (fused) saturated or
unsaturated carbocyclic or heterocyclic rings further substituted
by R.sub.3, (C.dbd.X)R.sub.3 and X(C.dbd.X)R.sub.3, including
epoxides and thioepoxides; J connected to I (or H), G (or F), K (or
L), M (or N) or S (or T) is a selection of C.sub.1-C.sub.8
disubstituted (fused) saturated and unsaturated carbocyclic or
heterocyclic rings further substituted by R.sub.3, (C.dbd.X)R.sub.3
and X(C.dbd.X)R.sub.3, including epoxides and thioepoxides; D (or
E) connected to B (or C) or G (or F); I (or H) connected to G (or
F); P (or O) connected to R (or Q) or M (or N); K (or L) connected
to N (or M) is a selection of C.sub.1-C.sub.8 disubstituted (fused)
saturated or unsaturated carbocyclic or heterocyclic rings
substituted by R.sub.3, (C.dbd.X)R.sub.3 and X(C.dbd.X)R.sub.3,
including epoxides and thioepoxides; B and C, D and E, R and Q, P
and O, I and H, G and F, K and L, M and N or S and T are .dbd.X
where X is selected from sulfur, oxygen, nitrogen, NR.sub.1R.sub.2,
and .dbd.CR.sub.1R.sub.2 14wherein: n is 0-10 atoms selected from
carbon, oxygen, nitrogen, sulfur, phosphorus, silicon, boron,
arsenic and selenium, wherein the ring defined by said atoms is
saturated or unsaturated, including epoxides and thioepoxides;
A'-T' are independently selected from hydrogen, R.sub.4, R.sub.5,
R.sub.6, F, Cl, Br, I, CN, COR.sub.4, CO.sub.2R.sub.4, OR.sub.4,
SR.sub.4, NR.sub.4R.sub.5, CONR.sub.4R.sub.5, N(.dbd.O).sub.2,
NR.sub.4OR.sub.5, ONR.sub.4R.sub.5, SOR.sub.4, SO.sub.2R.sub.4,
SO.sub.3R.sub.4, SONR.sub.4R.sub.5, S0.sub.2NR4R.sub.5,
S0.sub.3NR.sub.4R.sub.5, P(R.sub.4).sub.3,
P(.dbd.O)(R.sub.4).sub.3, Si(R.sub.4).sub.3, B(R.sub.4).sub.2,
(C.dbd.X)R.sub.6 or X(C.dbd.X)R.sub.6 where X is selected from
sulfur, oxygen and nitrogen; R.sub.4 and R.sub.5 are each
independently selected from C.sub.1-C.sub.20 alkyl (branched and/or
straight chained), C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and/or straight chained), C.sub.2-C.sub.10 alkynyl (branched and/or
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.4, SR.sub.4, NR.sub.4R.sub.5, N(.dbd.O).sub.2,
NR.sub.4OR.sub.5, ONR.sub.4R.sub.5, SOR.sub.4, SO.sub.2R.sub.4,
SO.sub.3R.sub.4, SONR.sub.4R.sub.5, SO.sub.2NR.sub.4R.sub.5,
SO.sub.3NR.sub.4R.sub.5, P(R.sub.4).sub.3,
P(.dbd.O)(R.sub.4).sub.3, Si(R.sub.4).sub.3,
B(P.sub.4).sub.2]alkyl; R.sub.6 is selected from R.sub.4, R.sub.5,
CN, COR.sub.4, CO.sub.2R.sub.4, OR.sub.4, SR.sub.4,
NR.sub.4R.sub.5, N(.dbd.O).sub.2, NR.sub.4OR.sub.5,
ONR.sub.4R.sub.5, SOR.sub.4, SO.sub.2R.sub.4, SO.sub.3R.sub.4,
SONR.sub.4R.sub.5, SO.sub.2NR.sub.4R.sub.5,
SO.sub.3NR.sub.4R.sub.5, P(R.sub.4).sub.3,
P(.dbd.O)(R.sub.4).sub.3, Si(R.sub.4).sub.3, B(R.sub.4).sub.2; E'
and R' or H' and O' is a C.sub.2-C.sub.8 saturated or unsaturated
carbocyclic or heterocyclic ring system further substituted by
R.sub.6, including epoxides and thioepoxides; O' connected to M'
(or N') or Q' (or P'); R' connected to Q' (or P') or S' (or T'); S'
(or T') connected to A' (or B'); A' (or B') connected to C' (or
D'); E' connected to C' (or D') or F' (or G'); H' connected to I';
I' connected to J'; J' connected to K'; K' connected to L'; L'
connected to M' (or N') are C.sub.1-C.sub.8 disubstituted (fused)
saturated or unsaturated carbocyclic or heterocyclic ring systems
further substituted by R.sub.6, (C.dbd.X)R.sub.6 and
X(C.dbd.X)R.sub.6, including epoxides and thioepoxides; A', B' and
C', D' and F', G' and M', N' and P', Q' and S', T' are .dbd.X where
X is selected from sulfur, oxygen, nitrogen, NR.sub.4R.sub.5,
(C.dbd.X)R.sub.6, X(C.dbd.X)R.sub.6, and .dbd.CR.sub.7R.sub.8;
R.sub.7 and R.sub.8 are each independently selected from R.sub.6,
(C.dbd.X)R.sub.6 and X(C.dbd.X)R.sub.6 15wherein: n is 0-10 atoms
selected from carbon, oxygen, nitrogen, sulfur, phosphorus,
silicon, boron, arsenic and selenium, wherein the ring defined by
said atoms is saturated or unsaturated, including epoxides and
thioepoxides; A.sup.1-T.sup.1 are independently selected from
hydrogen, R.sub.9, R.sub.10, R.sub.11, F, Cl, Br, I, CN, OR.sub.9,
SR.sub.9, NR.sub.9R.sub.10, N(.dbd.O).sub.2, NR.sub.9OR.sub.10,
ONR.sub.9R.sub.10, SOR.sub.9, SO.sub.2R.sub.9, SO.sub.3R.sub.9,
SONR.sub.9R.sub.10, SO.sub.2NR.sub.9R.sub.10,
SO.sub.3NR.sub.9R.sub.10, P(R.sub.9).sub.3,
P(.dbd.O)(R.sub.9).sub.3, Si(R.sub.9).sub.3, B(R.sub.9).sub.2,
(C.dbd.X)R.sub.11 or X(C.dbd.X)R.sub.11 where X is selected from
sulfur, oxygen and nitrogen; R.sub.9 and R.sub.10 are each
independently selected from C.sub.1-C.sub.20 alkyl (branched and
straight chained), C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and straight chained), C.sub.2-C.sub.10 alkynyl (branched and
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.9, SR.sub.9, NR.sub.9R.sub.10, N(.dbd.O).sub.2,
NR.sub.9OR.sub.10, ONR.sub.9R.sub.10, SOR.sub.9, SO.sub.2R.sub.9,
SO.sub.3R.sub.9, SONR.sub.9R.sub.10, SO.sub.2NR.sub.9R.sub.10,
SO.sub.3NR.sub.9R.sub.10, P(R.sub.9).sub.3,
P(.dbd.O)(R.sub.9).sub.3, Si(R.sub.9).sub.3,
B(R.sub.9).sub.2]alkyl; R.sub.11 is selected from R.sub.9,
R.sub.10, CN, COR.sub.9, CO.sub.2R.sub.9, OR.sub.9, SR.sub.9,
NR.sub.9R.sub.10, N(.dbd.O).sub.2, NR.sub.9OR.sub.10,
ONR.sub.9R.sub.10, SOR.sub.9, SO.sub.2R.sub.9, SO.sub.3R.sub.9,
SONR.sub.9R.sub.10, SO.sub.2NR.sub.9R.sub.10,
SO.sub.3NR.sub.9R.sub.10, P(R.sub.9).sub.3,
P(.dbd.O)(R.sub.9).sub.3, Si(R.sub.9).sub.3, B(R.sub.9).sub.2;
B.sup.1 and R.sup.1, E.sup.1 and .sup.1 and .sup.1 and M.sup.1 are
selected from a C.sub.2-C.sub.8 saturated or unsaturated
carbocyclic or heterocyclic ring system further substituted by
R.sub.11, including epoxides and thioepoxides; A.sup.1 (or .sup.1
connected to .sup.1 (or .sup.1) or T.sup.1 (or S.sup.1) B.sup.1
connected to .sup.1 (or .sup.1) or C.sup.1 (or D.sup.1) E.sup.1
connected to .sup.1 or C.sup.1 (or D.sup.1); .sup.1 connected to
.sup.1 (or F.sup.1); G.sup.1 (or H.sup.1) connected to .sup.1 (or
F.sup.1) or I.sup.1 (or J.sup.1); K.sup.1 (or L.sup.1) connected to
I.sup.1 (or J.sup.1) or M.sup.1; M.sup.1 connected to O.sup.1 (or
N.sup.1); .sup.1 connected O.sup.1 (or N.sup.1) or P.sup.1 (or
Q.sup.1); R.sup.1 connected P.sup.1 (or Q.sup.1) or S.sup.1 (or
T.sup.1) are C.sub.1-C.sub.8 disubstituted (fused) saturated or
unsaturated carbocyclic or heterocyclic ring systems further
substituted by R.sub.11, (C.dbd.X)R.sub.11 and X(C.dbd.X)R.sub.11,
including epoxides and thioepoxides; A.sup.1, and , and C.sup.1,
D.sup.1 and F.sup.1, and G.sup.1, H.sup.1 and I.sup.1, J.sup.1 and
K.sup.1, L.sup.1 and N.sup.1, O.sup.1 and P.sup.1, Q.sup.1 and
S.sup.1, T.sup.1 are .dbd.X where X is selected from sulfur,
oxygen, nitrogen, NR.sub.9R.sub.10, including (C.dbd.X)R.sub.11 and
X(C.dbd.X)R.sub.11, and .dbd.CR.sub.12R.sub.13; R.sub.12 and
R.sub.13 are independently selected from R.sub.11,
(C.dbd.X)R.sub.11 and X(C.dbd.X)R.sub.11 16wherein: n is 0-10 atoms
selected from carbon, oxygen, nitrogen, sulfur, phosphorus,
silicon, boron, arsenic and selenium, wherein the ring defined by
said atoms is saturated or unsaturated, including epoxides and
thioepoxides; A.sup.2-X.sup.2 are independently selected from
hydrogen, R.sub.14, R.sub.15, R.sub.16, F, Cl, Br, I, CN,
OR.sub.14, SR.sub.14, NR.sub.14R.sub.15, N(.dbd.O).sub.2,
NR.sub.14OR.sub.15, ONR.sub.14R.sub.15, SOR.sub.14,
SO.sub.2R.sub.14, SO.sub.3R.sub.14, SONR.sub.14R.sub.15,
SO.sub.2NR.sub.14R.sub.15, SO.sub.3NR.sub.14R.sub.15- ,
P(R.sub.14).sub.3, P(.dbd.O)(R.sub.14).sub.3, Si(R.sub.14).sub.3,
B(R.sub.14), (C.dbd.Y)R.sub.16 or Y(C.dbd.Y)R.sub.16 where Y is
selected from sulfur, oxygen and nitrogen; R.sub.14 and R.sub.15
are each independently selected from C.sub.1-C.sub.20 alkyl
(branched and/or straight chained), C.sub.1-C.sub.20 arylalkyl,
C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14
heteroaryl, C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl
(branched and/or straight chained), C.sub.2-C.sub.10 alkynyl
(branched and/or straight chained), C.sub.1-C.sub.10
heteroarylalkyl, C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10
haloalkyl, dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10
[CN, OR.sub.14, SR.sub.14, NR.sub.14R.sub.10, N(.dbd.O).sub.2,
NR.sub.14OR.sub.15, ONR.sub.14R.sub.15, SOR.sub.14,
SO.sub.2R.sub.14, SO.sub.3R.sub.14, SONR.sub.14R.sub.15,
SO.sub.2NR.sub.14R.sub.15, SO.sub.3NR.sub.14R.sub.15,
P(R.sub.14).sub.3, P(.dbd.O)(R.sub.14).sub.3, Si(R.sub.14).sub.3,
B(R.sub.14).sub.2]alkyl; R.sub.16 is selected from R.sub.14,
R.sub.15, CN, COR.sub.14, CO.sub.2R.sub.15, OR.sub.14, SR.sub.14,
NR.sub.14R.sub.15, N(.dbd.O).sub.2, NR.sub.14OR.sub.15,
ONR.sub.14R.sub.15, SOR.sub.14, SO.sub.2R.sub.14, SO.sub.3R.sub.14,
SONR.sub.14R.sub.15, SO.sub.2NR.sub.14R.sub.15,
SO.sub.3NR.sub.14R.sub.15, P(R.sub.14).sub.3,
P(.dbd.O)(R.sub.14).sub.3, Si(R.sub.14).sub.3, B(R.sub.14).sub.2;
E.sup.2 and V.sup.2, H.sup.2 and S.sup.2, and I.sup.2 and P.sup.2
are C.sub.2-C.sub.8 saturated or unsaturated carbocyclic or
heterocyclic ring system further substituted by R.sub.16, including
epoxides and thioepoxides; A.sup.2 (or B.sup.2) connected to
C.sup.2 (or D.sup.2) or W.sup.2 (or X.sup.2); E.sup.2 connected to
C.sup.2 (or D.sup.2) or F.sup.2 (or G.sup.2); H.sup.2 connected to
F.sup.2 (or G.sup.2) or I.sup.2; I.sup.2 connected to J.sup.2 (or
K.sup.2); L.sup.2 (or M.sup.2) connected to J.sup.2 (or K.sup.2) or
N.sup.2 (or O.sup.2); R.sup.2 (or Q.sup.2) connected to P.sup.2 or
S.sup.2; V.sup.2 connected to U.sup.2 (or T.sup.2) or W.sup.2 (or
X.sup.2) are C.sub.1-C.sub.8 disubstituted (fused) saturated or
unsaturated carbocyclic or heterocyclic ring systems further
substituted by R.sub.16, (C.dbd.Y)R.sub.16 and Y(C.dbd.Y)R.sub.16,
including epoxides and thioepoxides; A.sup.2, B.sup.2; C.sup.2,
D.sup.2; F.sup.2, G.sup.2; J.sup.2, K.sup.2; L.sup.2, M.sup.2;
N.sup.2, O.sup.2; Q.sup.2, R.sup.2; U.sup.2, T.sup.2 and X.sup.2,
W.sup.2 are .dbd.Y where Y is selected from sulfur, oxygen,
nitrogen, NR.sub.14R.sub.15 and .dbd.CR.sub.17R.sub.18; R.sub.17
and R.sub.18 are independently selected from R.sub.16,
(C.dbd.Y)R.sub.16 and Y(C.dbd.Y)R.sub.16 17wherein: n is 0-10 atoms
selected from carbon, oxygen, nitrogen, sulfur, phosphorus,
silicon, boron, arsenic and selenium, wherein the ring defined by
said atoms is saturated or unsaturated, including epoxides and
thioepoxides; A.sup.3-Z.sup.3 are independently selected from
hydrogen, R.sub.19, R.sub.20, R.sub.21, F, Cl, Br, I, CN,
OR.sub.19, SR.sub.19, NR.sub.19R.sub.20, N(.dbd.O).sub.2,
NR.sub.19OR.sub.20, ONR.sub.19R.sub.20, SOR.sub.19,
SO.sub.2R.sub.19, SO.sub.3R.sub.19, SONR.sub.19R.sub.20,
SO.sub.2NR.sub.19R.sub.20, SO.sub.3NR.sub.19R.sub.20,
P(R,.sub.9).sub.3, P(O)(R.sub.19).sub.3, Si(R.sub.19).sub.3,
B(R.sub.19).sub.2, (C.dbd..O slashed.)R.sub.21 or .O
slashed.(C.dbd..O slashed.)R.sub.21 where .O slashed. is sullur,
oxygen and nitrogen; R.sub.19 and R.sub.20 are each independently
selected from C.sub.1-C.sub.20 alkyl (branched and/or straight
chained), C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8 cycloalkyl,
C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and/or straight chained), C.sub.2-C.sub.10 alkynyl (branched and/or
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.19, SR.sub.19, NR.sub.19R.sub.20, N(.dbd.O).sub.2,
NR.sub.19OR.sub.20, ONR.sub.19R.sub.20, SOR.sub.19,
SO.sub.2R.sub.19, SO.sub.3R.sub.19, SONR.sub.19R.sub.20,
SO.sub.2NR.sub.19R.sub.20, SO.sub.3NR.sub.19R.sub.20,
P(R.sub.19).sub.3, P(.dbd.O)(R.sub.19).sub.3, Si(R.sub.19).sub.3,
B(R.sub.19).sub.2]alkyl; R.sub.21 is selected from R.sub.19,
R.sub.20, CN, COR.sub.19, CO.sub.2R.sub.19, OR.sub.19, SR.sub.19,
NR.sub.19R.sub.20, N(.dbd.O).sub.2, NR.sub.19OR.sub.20,
ONR.sub.19R.sub.20, SOR.sub.19, SO.sub.2R.sub.19, SO.sub.3R.sub.19,
SONR.sub.19R.sub.20, SO.sub.2NR.sub.19R.sub.20,
SO.sub.3NR.sub.19R.sub.20, P(R.sub.19).sub.3,
P(.dbd.O)(R.sub.19).sub.3, Si(R.sub.19).sub.3, B(R.sub.19).sub.2;
D.sup.3 connected to X.sup.3 is a C.sub.2-C.sub.8 saturated or
unsaturated carbocyclic or heterocyclic ring system further
substituted by R.sub.21, including epoxides and thioepoxides;
A.sup.3 (or .sup.3) connected to B.sup.3 (or C.sup.3) or E.sup.3
(or X); D.sup.3 connected to B.sup.3 (or C.sup.3) or E.sup.3 (or
F.sup.3); G.sup.3 (or H.sup.3) connected to E.sup.3 (or F.sup.3) or
I.sup.3 (or J.sup.3); L.sup.3 (or K.sup.3) connected to I.sup.3 (or
J.sup.3) or M.sup.3 (or N.sup.3); O.sup.3 (or .sup.3) connected to
N.sup.3 (or M.sup.3) or P.sup.3 (or Q.sup.3). S.sup.3 (or R.sup.3)
connected to Q.sup.3 (or P.sup.3) or U.sup.3 (or T.sup.3). W.sup.3
(or V.sup.3) connected to U.sup.3 (or T.sup.3) or X.sup.3; X.sup.3
connected to Y.sup.3 (or Z.sup.3) are C.sub.1-C.sub.8 disubstituted
(fused) saturated or unsaturated carbocyclic or heterocyclic ring
systems further substituted by R.sub.21, (C.dbd..O
slashed.)R.sub.21 and .O slashed. (C.dbd..O slashed.)R.sub.21,
including epoxides and thioepoxides; A.sup.3, .sup.3; B.sup.3,
C.sup.3; E.sup.3, F.sup.3; G.sup.3, H.sup.3; I.sup.3, J.sup.3;
K.sup.3, L.sup.3; M.sup.3, N.sup.3; O.sup.3, .sup.3; Q.sup.3,
P.sup.3, S.sup.3, R.sup.3, U.sup.3, T.sup.3, W.sup.3, V.sup.3, and
Z.sup.3, Y.sup.3 are .dbd..O slashed. where .O slashed. is selected
from sulfur, oxygen, nitrogen, NR.sub.19R.sub.20, and
.dbd.CR.sub.22R.sub.23; and R.sub.22 and R.sub.23 are selected from
R.sub.21, (C.dbd..O slashed.)R.sub.21 and .O slashed.(C.dbd..O
slashed.)R.sub.21; and which chemical agent or derivative or
chemical analogue thereof is capable of modulating PKC activity,
PKC-gene expression or PKC enzyme turnover, said method comprising
administering to said subject an expression facilitating amount of
said chemical agent or derivative or chemical analogue thereof for
a time and under conditions sufficient to facilitate the expression
of said genetic sequence to thereby ameliorate one or more
symptoms
associated with said condition or disorder.
59. A method according to claim 58 wherein the chemical agent is
represented by the general formula (VI): 18wherein: R.sub.24,
R.sub.25 and R.sub.26 are independently selected from hydrogen,
R.sub.27, R.sub.28, F, Cl, Br, I, CN, OR.sub.27, SR.sub.27,
NR.sub.27R.sub.28, N(.dbd.O).sub.2, NR.sub.27OR.sub.28,
ONR.sub.27R.sub.28, SOR.sub.27, SO.sub.2R.sub.27, SO.sub.3R.sub.27,
SONR.sub.27R.sub.28, SO.sub.2NR.sub.27R.sub.28,
SO.sub.3NR.sub.27R.sub.28, P(R.sub.27).sub.3,
P(.dbd.O)(R.sub.27).sub.3, Si(R.sub.27).sub.3, B(R.sub.27).sub.2,
(C.dbd.X)R.sub.29 or X(C.dbd.X)R.sub.29 where X is selected from
sulfur, oxygen and nitrogen; R.sub.27 and R.sub.28 are each
independently selected from C.sub.1-C.sub.20 alkyl (branched and/or
straight chained), C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and/or straight chained), C.sub.2-C.sub.10 alkynyl (branched and/or
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.27, SR.sub.27, NR.sub.27R.sub.28, N(.dbd.O).sub.2,
NR.sub.27OR.sub.28, ONR.sub.27R.sub.28, SOR.sub.27,
SO.sub.2R.sub.27, SO.sub.3R.sub.27, SONR.sub.27R.sub.28,
SO.sub.2NR.sub.27R.sub.28, SO.sub.3NR.sub.27R.sub.28,
P(R.sub.27).sub.3, P(.dbd.O)(R.sub.27).sub.3, Si(R.sub.27).sub.3,
B(R.sub.27).sub.2]alkyl; R.sub.29 is selected from R.sub.27,
R.sub.28, CN, COR.sub.27, CO.sub.2R.sub.27, OR.sub.27, SR.sub.27,
NR.sub.27R.sub.28, N(.dbd.O).sub.2, NR.sub.27OR.sub.28,
ONR.sub.27R.sub.28, SOR.sub.27, S0.sub.2R.sub.27, SO.sub.3R.sub.27,
SONR.sub.27R.sub.28, SO.sub.2NR.sub.27R.sub.28,
SO.sub.3NR.sub.27R.sub.28, P(R.sub.27).sub.3,
P(.dbd.O)(R.sub.27).sub.3, Si(R.sub.27).sub.3,
B(R.sub.27).sub.2.
60. A method of modulating the expression of a genetic sequence by
the administration of a chemical agent obtainable from a plant of
the Euphorbiaceae family or a derivative or chemical analogue
thereof which chemical agent is a macrocyclic diterpene selected
from compounds of the ingenane, pepluane and jatrophane familes and
which chemical agent or derivative or chemical analogue is
represented by any one of the general formulae (I)-(V): 19wherein:
n is 0-10 atoms selected from carbon, oxygen, nitrogen, sulfur,
phosphorus, silicon, boron, arsenic and selenium, wherein the ring
defined by said atoms is saturated or unsaturated, including
epoxides and thioepoxides; A-T are independently selected from
hydrogen, R.sub.1, R.sub.2, R.sub.3, F, Cl, Br, I, CN, OR.sub.1,
SR.sub.1, NR.sub.1R.sub.2, N(.dbd.O).sub.2, NR.sub.1OR.sub.2,
ONR.sub.1R.sub.2, SOR.sub.1, SO.sub.2R.sub.1, SO.sub.3R.sub.1,
SONR.sub.1R.sub.2, SO.sub.2NR.sub.1R.sub.2,
SO.sub.3NR.sub.1R.sub.2, P(R.sub.1).sub.3,
P(.dbd.O)(R.sub.1).sub.3, Si(R.sub.1).sub.3, B(R.sub.1).sub.2,
(C.dbd.X)R.sub.3 or X(C.dbd.X)R.sub.3 where X is selected from
sulfur, oxygen and nitrogen; R.sub.1 and R.sub.2 are each
independently selected from C.sub.1-C.sub.20 alkyl (branched and/or
straight chained), C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and/or straight chained), C.sub.2-C.sub.10 alkynyl (branched and/or
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.1, SR.sub.1, NR.sub.1R.sub.2, N(.dbd.O).sub.2,
NR.sub.1OR.sub.2, ONR.sub.1R.sub.2, SOR.sub.1, SO.sub.2R.sub.1,
SO.sub.3R.sub.1, SONR.sub.1R.sub.2, SO.sub.2NR.sub.1R.sub.2,
SO.sub.3NR.sub.1R.sub.2, P(R.sub.1).sub.3,
P(.dbd.O)(R.sub.1).sub.3, Si(R.sub.1).sub.3,
B(R.sub.1).sub.2]alkyl; R.sub.3 is selected from R.sub.1, R.sub.2,
CN, COR.sub.1, CO.sub.2R.sub.1, OR.sub.1, SR.sub.1,
NR.sub.1R.sub.2, N(.dbd.O).sub.2, NR.sub.1OR.sub.2,
ONR.sub.1R.sub.2, SOR.sub.1, SO.sub.2R.sub.1, SO.sub.3R.sub.1,
SONR.sub.1R.sub.2, SO.sub.2NR.sub.1R.sub.2,
SO.sub.3NR.sub.1R.sub.2, P(R.sub.1).sub.3,
P(.dbd.O)(R.sub.1).sub.3, Si(R.sub.1).sub.3, B(R.sub.1).sub.2; A
connected to B (or C), D (or E, R (or Q), P (or O) or S (or T) is a
selection of C.sub.1-C.sub.8 disubstituted (fused) saturated or
unsaturated carbocyclic or heterocyclic rings further substituted
by R.sub.3, (C.dbd.X)R.sub.3 and X(C.dbd.X)R.sub.3, including
epoxides and thioepoxides; J connected to I (or H), G (or F), K (or
L), M (or N) or S (or T) is a selection of C.sub.1-C.sub.8
disubstituted (fused) saturated and unsaturated carbocyclic or
heterocyclic rings further substituted by R.sub.3, (C.dbd.X)R.sub.3
and X(C.dbd.X)R.sub.3, including epoxides and thioepoxides; D (or
E) connected to B (or C) or G (or F); I (or H) connected to G (or
F); P (or O) connected to R (or Q) or M (or N); K (or L) connected
to N (or M) is a selection of C.sub.1-C.sub.8 disubstituted (fused)
saturated or unsaturated carbocyclic or heterocyclic rings
substituted by R.sub.3, (C.dbd.X)R.sub.3 and X(C.dbd.X)R.sub.3,
including epoxides and thioepoxides; B and C, D and E, R and Q, P
and O, I and H, G and F, K and L, M and N or S and T are .dbd.X
where X is selected from sulfur, oxygen, nitrogen, NR.sub.1R.sub.2,
and .dbd.CR.sub.1R.sub.2 20wherein: n is 0-10 atoms selected from
carbon, oxygen, nitrogen, sulfur, phosphorus, silicon, boron,
arsenic and selenium, wherein the ring defined by said atoms is
saturated or unsaturated, including epoxides and thioepoxides;
A'-T' are independently selected from hydrogen, R.sub.4, R.sub.5,
R.sub.6, F, Cl, Br, I, CN, COR.sub.4, CO.sub.2R.sub.4, OR.sub.4,
SR.sub.4, NR.sub.4R.sub.5, CONR.sub.4R.sub.5, N(.dbd.O).sub.2,
NR.sub.4OR.sub.5, ONR.sub.4R.sub.5, SOR.sub.4, SO.sub.2R.sub.4,
SO.sub.3R.sub.4, SONR.sub.4R.sub.5, S0.sub.2NR4R.sub.5,
S0.sub.3NR.sub.4R.sub.5, P(R.sub.4).sub.3,
P(.dbd.O)(R.sub.4).sub.3, Si(R.sub.4).sub.3, B(R.sub.4).sub.2,
(C.dbd.X)R.sub.6 or X(C.dbd.X)R.sub.6 where X is selected from
sulfur, oxygen and nitrogen; R.sub.4 and R.sub.5 are each
independently selected from C.sub.1-C.sub.20 alkyl (branched and/or
straight chained), C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and/or straight chained), C.sub.2-C.sub.10 alkynyl (branched and/or
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.4, SR.sub.4, NR.sub.4R.sub.5, N(.dbd.O).sub.2,
NR.sub.4OR.sub.5, ONR.sub.4R.sub.5, SOR.sub.4, SO.sub.2R.sub.4,
SO.sub.3R.sub.4, SONR.sub.4R.sub.5, SO.sub.2NR.sub.4R.sub.5,
SO.sub.3NR.sub.4R.sub.5, P(R.sub.4).sub.3,
P(.dbd.O)(R.sub.4).sub.3, Si(R.sub.4).sub.3,
B(P.sub.4).sub.2]alkyl; R.sub.6 is selected from R.sub.4, R.sub.5,
CN, COR.sub.4, CO.sub.2R.sub.4, OR.sub.4, SR.sub.4,
NR.sub.4R.sub.5, N(.dbd.O).sub.2, NR.sub.4OR.sub.5,
ONR.sub.4R.sub.5, SOR.sub.4, SO.sub.2R.sub.4, SO.sub.3R.sub.4,
SONR.sub.4R.sub.5, SO.sub.2NR.sub.4R.sub.5,
SO.sub.3NR.sub.4R.sub.5, P(R.sub.4).sub.3,
P(.dbd.O)(R.sub.4).sub.3, Si(R.sub.4).sub.3, B(R.sub.4).sub.2; E'
and R' or H' and O' is a C.sub.2-C.sub.8 saturated or unsaturated
carbocyclic or heterocyclic ring system further substituted by
R.sub.6, including epoxides and thioepoxides; O' connected to M'
(or N') or Q' (or P'); R' connected to Q' (or P') or S' (or T'); S'
(or T') connected to A' (or B'); A' (or B') connected to C' (or
D'); E' connected to C' (or D') or F' (or G'); H' connected to I';
I' connected to J'; J' connected to K'; K' connected to L'; L'
connected to M' (or N') are C.sub.1-C.sub.8 disubstituted (fused)
saturated or unsaturated carbocyclic or heterocyclic ring systems
further substituted by R.sub.6, (C.dbd.X)R.sub.6 and
X(C.dbd.X)R.sub.6, including epoxides and thioepoxides; A', B' and
C', D' and F', G' and M', N' and P', Q' and S', T' are .dbd.X where
X is selected from sulfur, oxygen, nitrogen, NR.sub.4R.sub.5,
(C.dbd.X)R.sub.6, X(C.dbd.X)R.sub.6, and .dbd.CR.sub.7R.sub.8;
R.sub.7 and R.sub.8 are each independently selected from R.sub.6,
(C.dbd.X)R.sub.6 and X(C.dbd.X)R.sub.6 21wherein: n is 0-10 atoms
selected from carbon, oxygen, nitrogen, sulfur, phosphorus,
silicon, boron, arsenic and selenium, wherein the ring defined by
said atoms is saturated or unsaturated, including epoxides and
thioepoxides; A.sup.1-T.sup.1 are independently selected from
hydrogen, R.sub.9, R.sub.10, R.sub.11, F, Cl, Br, I, CN, OR.sub.9,
SR.sub.9, NR.sub.9R.sub.10, N(.dbd.O).sub.2, NR.sub.9OR.sub.10,
ONR.sub.9R.sub.10, SOR.sub.9, SO.sub.2R.sub.9, SO.sub.3R.sub.9,
SONR.sub.9R.sub.10, SO.sub.2NR.sub.9R.sub.10,
SO.sub.3NR.sub.9R.sub.10, P(R.sub.9).sub.3,
P(.dbd.O)(R.sub.9).sub.3, Si(R.sub.9).sub.3, B(R.sub.9).sub.2,
(C.dbd.X)R.sub.11 or X(C.dbd.X)R.sub.11 where X is selected from
sulfur, oxygen and nitrogen; R.sub.9 and R.sub.10 are each
independently selected from C.sub.1-C.sub.20 alkyl (branched and
straight chained), C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and straight chained), C.sub.2-C.sub.10 alkynyl (branched and
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.9, SR.sub.9, NR.sub.9R.sub.10, N(.dbd.O).sub.2,
NR.sub.9OR.sub.10, ONR.sub.9R.sub.10, SOR.sub.9, SO.sub.2R.sub.9,
SO.sub.3R.sub.9, SONR.sub.9R.sub.10, SO.sub.2NR.sub.9R.sub.10,
SO.sub.3NR.sub.9R.sub.10, P(R.sub.9).sub.3,
P(.dbd.O)(R.sub.9).sub.3, Si(R.sub.9).sub.3,
B(R.sub.9).sub.2]alkyl; R.sub.11 is selected from R.sub.9,
R.sub.10, CN, COR.sub.9, CO.sub.2R.sub.9, OR.sub.9, SR.sub.9,
NR.sub.9R.sub.10, N(.dbd.O).sub.2, NR.sub.9OR.sub.10,
ONR.sub.9R.sub.10, SOR.sub.9, SO.sub.2R.sub.9, SO.sub.3R.sub.9,
SONR.sub.9R.sub.10, SO.sub.2NR.sub.9R.sub.10,
SO.sub.3NR.sub.9R.sub.10, P(R.sub.9).sub.3,
P(.dbd.O)(R.sub.9).sub.3, Si(R.sub.9).sub.3, B(R.sub.9).sub.2;
B.sup.1 and R.sup.1, E.sup.1 and .sup.1 and .sup.1 and M.sup.1 are
selected from a C.sub.2-C.sub.8 saturated or unsaturated
carbocyclic or heterocyclic ring system further substituted by
R.sub.11, including epoxides and thioepoxides; A.sup.1 (or .sup.1
connected to .sup.1 (or .sup.1) or T.sup.1 (or S.sup.1) B.sup.1
connected to .sup.1 (or .sup.1) or C.sup.1 (or D.sup.1) E.sup.1
connected to .sup.1 or C.sup.1 (or D.sup.1); .sup.1 connected to
.sup.1 (or F.sup.1); G.sup.1 (or H.sup.1) connected to .sup.1 (or
F.sup.1) or I.sup.1 (or J.sup.1); K.sup.1 (or L.sup.1) connected to
I.sup.1 (or J.sup.1) or M.sup.1; M.sup.1 connected to O.sup.1 (or
N.sup.1); .sup.1 connected O.sup.1 (or N.sup.1) or P.sup.1 (or
Q.sup.1); R.sup.1 connected P.sup.1 (or Q.sup.1) or S.sup.1 (or
T.sup.1) are C.sub.1-C.sub.8 disubstituted (fused) saturated or
unsaturated carbocyclic or heterocyclic ring systems further
substituted by R.sub.11, (C.dbd.X)R.sub.11 and X(C.dbd.X)R.sub.11,
including epoxides and thioepoxides; A.sup.1, and , and C.sup.1,
D.sup.1 and F.sup.1, and G.sup.1, H.sup.1 and I.sup.1, J.sup.1 and
K.sup.1, L.sup.1 and N.sup.1, O.sup.1 and P.sup.1, Q.sup.1 and
S.sup.1, T.sup.1 are .dbd.X where X is selected from sulfur,
oxygen, nitrogen, NR.sub.9R.sub.10, including (C.dbd.X)R.sub.11 and
X(C.dbd.X)R.sub.11, and .dbd.CR.sub.12R.sub.13; R.sub.12 and
R.sub.13 are independently selected from R.sub.11,
(C.dbd.X)R.sub.11 and X(C.dbd.X)R.sub.11 22wherein: n is 0-10 atoms
selected from carbon, oxygen, nitrogen, sulfur, phosphorus,
silicon, boron, arsenic and selenium, wherein the ring defined by
said atoms is saturated or unsaturated, including epoxides and
thioepoxides; A.sup.2-X.sup.2 are independently selected from
hydrogen, R.sub.14, R.sub.15, R.sub.16, F, Cl, Br, I, CN,
OR.sub.14, SR.sub.14, NR.sub.14R.sub.15, N(.dbd.O).sub.2,
NR.sub.14OR.sub.15, ONR.sub.14R.sub.15, SOR.sub.14,
SO.sub.2R.sub.14, SO.sub.3R.sub.14, SONR.sub.14R.sub.15,
SO.sub.2NR.sub.14R.sub.15, SO.sub.3NR.sub.14R.sub.15- ,
P(R.sub.14).sub.3, P(.dbd.O)(R.sub.14).sub.3, Si(R.sub.14).sub.3,
B(R.sub.14), (C.dbd.Y)R.sub.16 or Y(C.dbd.Y)R.sub.16 where Y is
selected from sulfur, oxygen and nitrogen; R.sub.14 and R.sub.15
are each independently selected from C.sub.1-C.sub.20 alkyl
(branched and/or straight chained), C.sub.1-C.sub.20 arylalkyl,
C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14
heteroaryl, C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl
(branched and/or straight chained), C.sub.2-C.sub.10 alkynyl
(branched and/or straight chained), C.sub.1-C.sub.10
heteroarylalkyl, C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10
haloalkyl, dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10
[CN, OR.sub.14, SR.sub.14, NR.sub.14R.sub.10, N(.dbd.O).sub.2,
NR.sub.14OR.sub.15, ONR.sub.14R.sub.15, SOR.sub.14,
SO.sub.2R.sub.14, SO.sub.3R.sub.14, SONR.sub.14R.sub.15,
SO.sub.2NR.sub.14R.sub.15, SO.sub.3NR.sub.14R.sub.15,
P(R.sub.14).sub.3, P(.dbd.O)(R.sub.14).sub.3, Si(R.sub.14).sub.3,
B(R.sub.14).sub.2]alkyl; R.sub.16 is selected from R.sub.14,
R.sub.15, CN, COR.sub.14, CO.sub.2R.sub.15, OR.sub.14, SR.sub.14,
NR.sub.14R.sub.15, N(.dbd.O).sub.2, NR.sub.14OR.sub.15,
ONR.sub.14R.sub.15, SOR.sub.14, SO.sub.2R.sub.14, SO.sub.3R.sub.14,
SONR.sub.14R.sub.15, SO.sub.2NR.sub.14R.sub.15,
SO.sub.3NR.sub.14R.sub.15, P(R.sub.14).sub.3,
P(.dbd.O)(R.sub.14).sub.3, Si(R.sub.14).sub.3, B(R.sub.14).sub.2;
E.sup.2 and V.sup.2, H.sup.2 and S.sup.2, and I.sup.2 and P.sup.2
are C.sub.2-C.sub.8 saturated or unsaturated carbocyclic or
heterocyclic ring system further substituted by R.sub.16, including
epoxides and thioepoxides; A.sup.2 (or B.sup.2) connected to
C.sup.2 (or D.sup.2) or W.sup.2 (or X.sup.2); E.sup.2 connected to
C.sup.2 (or D.sup.2) or F.sup.2 (or G.sup.2); H.sup.2 connected to
F.sup.2 (or G.sup.2) or I.sup.2; I.sup.2 connected to J.sup.2 (or
K.sup.2); L.sup.2 (or M.sup.2) connected to J.sup.2 (or K.sup.2) or
N.sup.2 (or O.sup.2); R.sup.2 (or Q.sup.2) connected to P.sup.2 or
S.sup.2; V.sup.2 connected to U.sup.2 (or T.sup.2) or W.sup.2 (or
X.sup.2) are C.sub.1-C.sub.8 disubstituted (fused) saturated or
unsaturated carbocyclic or heterocyclic ring systems further
substituted by R.sub.16, (C.dbd.Y)R.sub.16 and Y(C.dbd.Y)R.sub.16,
including epoxides and thioepoxides; A.sup.2, B.sup.2; C.sup.2,
D.sup.2; F.sup.2, G.sup.2; J.sup.2, K.sup.2; L.sup.2, M.sup.2;
N.sup.2, O.sup.2; Q.sup.2, R.sup.2; U.sup.2, T.sup.2 and X.sup.2,
W.sup.2 are .dbd.Y where Y is selected from sulfur, oxygen,
nitrogen, NR.sub.14R.sub.15 and .dbd.CR.sub.17R.sub.18; R.sub.17
and R.sub.18 are independently selected from R.sub.16,
(C.dbd.Y)R.sub.16 and Y(C.dbd.Y)R.sub.16 23wherein: n is 0-10 atoms
selected from carbon, oxygen, nitrogen, sulfur, phosphorus,
silicon, boron, arsenic and selenium, wherein the ring defined by
said atoms is saturated or unsaturated, including epoxides and
thioepoxides; A.sup.3-Z.sup.3 are independently selected from
hydrogen, R.sub.19, R.sub.20, R.sub.21, F, Cl, Br, I, CN,
OR.sub.19, SR.sub.19, NR.sub.19R.sub.20, N(.dbd.O).sub.2,
NR.sub.19OR.sub.20, ONR.sub.19R.sub.20, SOR.sub.19,
SO.sub.2R.sub.19, SO.sub.3R.sub.19, SONR.sub.19R.sub.20,
SO.sub.2NR.sub.19R.sub.20, SO.sub.3NR.sub.19R.sub.20,
P(R,.sub.9).sub.3, P(O)(R.sub.19).sub.3, Si(R.sub.19).sub.3,
B(R.sub.19).sub.2, (C.dbd..O slashed.)R.sub.21 or .O
slashed.(C.dbd..O slashed.)R.sub.21 where .O slashed. is sullur,
oxygen and nitrogen; R.sub.19 and R.sub.20 are each independently
selected from C.sub.1-C.sub.20 alkyl (branched and/or straight
chained), C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8 cycloalkyl,
C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and/or straight chained), C.sub.2-C.sub.10 alkynyl (branched and/or
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.19, SR.sub.19, NR.sub.19R.sub.20, N(.dbd.O).sub.2,
NR.sub.19OR.sub.20, ONR.sub.19R.sub.20, SOR.sub.19,
SO.sub.2R.sub.19, SO.sub.3R.sub.19, SONR.sub.19R.sub.20,
SO.sub.2NR.sub.19R.sub.20, SO.sub.3NR.sub.19R.sub.20,
P(R.sub.19).sub.3, P(.dbd.O)(R.sub.19).sub.3, Si(R.sub.19).sub.3,
B(R.sub.19).sub.2]alkyl; R.sub.21 is selected from R.sub.19,
R.sub.20, CN, COR.sub.19, CO.sub.2R.sub.19, OR.sub.19, SR.sub.19,
NR.sub.19R.sub.20, N(.dbd.O).sub.2, NR.sub.19OR.sub.20,
ONR.sub.19R.sub.20, SOR.sub.19, SO.sub.2R.sub.19, SO.sub.3R.sub.19,
SONR.sub.19R.sub.20, SO.sub.2NR.sub.19R.sub.20,
SO.sub.3NR.sub.19R.sub.20, P(R.sub.19).sub.3,
P(.dbd.O)(R.sub.19).sub.3, Si(R.sub.19).sub.3, B(R.sub.19).sub.2;
D.sup.3 connected to X.sup.3 is a C.sub.2-C.sub.8 saturated or
unsaturated carbocyclic or heterocyclic ring system further
substituted by R.sub.21, including epoxides and thioepoxides;
A.sup.3 (or .sup.3) connected to B.sup.3 (or C.sup.3) or E.sup.3
(or X); D.sup.3 connected to B.sup.3 (or C.sup.3) or E.sup.3 (or
F.sup.3); G.sup.3 (or H.sup.3) connected to E.sup.3 (or F.sup.3) or
I.sup.3 (or J.sup.3); L.sup.3 (or K.sup.3) connected to I.sup.3 (or
J.sup.3) or M.sup.3 (or N.sup.3); O.sup.3 (or .sup.3) connected to
N.sup.3 (or M.sup.3) or P.sup.3 (or Q.sup.3). S.sup.3 (or R.sup.3)
connected to Q.sup.3 (or P.sup.3) or U.sup.3 (or T.sup.3). W.sup.3
(or V.sup.3) connected to U.sup.3 (or T.sup.3) or X.sup.3; X.sup.3
connected to Y.sup.3 (or Z.sup.3) are C.sub.1-C.sub.8 disubstituted
(fused) saturated or unsaturated carbocyclic or heterocyclic ring
systems further substituted by R.sub.21, (C.dbd..O
slashed.)R.sub.21 and .O slashed. (C.dbd..O slashed.)R.sub.21,
including epoxides and thioepoxides; A.sup.3, .sup.3; B.sup.3,
C.sup.3; E.sup.3, F.sup.3; G.sup.3, H.sup.3; I.sup.3, J.sup.3;
K.sup.3, L.sup.3; M.sup.3, N.sup.3; O.sup.3, .sup.3; Q.sup.3,
P.sup.3, S.sup.3, R.sup.3, U.sup.3, T.sup.3, W.sup.3, V.sup.3, and
Z.sup.3, Y.sup.3 are .dbd..O slashed. where .O slashed. is selected
from sulfur, oxygen, nitrogen, NR.sub.19R.sub.20, and
.dbd.CR.sub.22R.sub.23; and R.sub.22 and R.sub.23 are selected from
R.sub.21, (C.dbd..O slashed.)R.sub.21 and .O slashed.(C.dbd..O
slashed.)R.sub.21; and which chemical agent or derivative or
chemical analogue thereof is capable of modulating PKC activity,
PKC-gene expression or PKC enzyme turnover and wherein said
chemical agent or its derivatives or chemical analogues is
administered for a time and under conditions sufficient to
facilitate the expression of said genetic sequence.
61. A method according to claim 60 wherein the chemical agent is
represented by the general formula (VI): 24wherein: R.sub.24,
R.sub.25 and R.sub.26 are independently selected from hydrogen,
R.sub.27, R.sub.28, F, Cl, Br, I, CN, OR.sub.27, SR.sub.27,
NR.sub.27R.sub.28, N(.dbd.O).sub.2, NR.sub.27OR.sub.28,
ONR.sub.27R.sub.28, SOR.sub.27, SO.sub.2R.sub.27, SO.sub.3R.sub.27,
SONR.sub.27R.sub.28, SO.sub.2NR.sub.27R.sub.28,
SO.sub.3NR.sub.27R.sub.28, P(R.sub.27).sub.3,
P(.dbd.O)(R.sub.27).sub.3, Si(R.sub.27).sub.3, B(R.sub.27).sub.2,
(C.dbd.X)R.sub.29 or X(C.dbd.X)R.sub.29 where X is selected from
sulfur, oxygen and nitrogen; R.sub.27 and R.sub.28 are each
independently selected from C.sub.1-C.sub.20 alkyl (branched and/or
straight chained), C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and/or straight chained), C.sub.2-C.sub.10 alkynyl (branched and/or
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.27, SR.sub.27, NR.sub.27R.sub.28, N(.dbd.O).sub.2,
NR.sub.27OR.sub.28, ONR.sub.27R.sub.28, SOR.sub.27,
SO.sub.2R.sub.27, SO.sub.3R.sub.27, SONR.sub.27R.sub.28,
SO.sub.2NR.sub.27R.sub.28, SO.sub.3NR.sub.27R.sub.28,
P(R.sub.27).sub.3, P(.dbd.O)(R.sub.27).sub.3, Si(R.sub.27).sub.3,
B(R.sub.27).sub.2]alkyl; R.sub.29 is selected from R.sub.27,
R.sub.28, CN, COR.sub.27, CO.sub.2R.sub.27, OR.sub.27, SR.sub.27,
NR.sub.27R.sub.28, N(.dbd.O).sub.2, NR.sub.27OR.sub.28,
ONR.sub.27R.sub.28, SOR.sub.27, S0.sub.2R.sub.27, SO.sub.3R.sub.27,
SONR.sub.27R.sub.28, SO.sub.2NR.sub.27R.sub.28,
SO.sub.3NR.sub.27R.sub.28, P(R.sub.27).sub.3,
P(.dbd.O)(R.sub.27).sub.3, Si(R.sub.27).sub.3,
B(R.sub.27).sub.2.
62. A method of stimulating the activation or function of a
promoter by the administration of a chemical agent obtainable from
a plant of the Euphorbiaceae family or a derivative or chemical
analogue thereof which chemical agent is a macrocyclic diterpene
selected from compounds of the ingenane, pepluane and jatrophane
families and which chemical agent or derivative or chemical
analogue is represented by any one of the general formulae (I)-(V):
25wherein: n is 0-10 atoms selected from carbon, oxygen, nitrogen,
sulfur, phosphorus, silicon, boron, arsenic and selenium, wherein
the ring defined by said atoms is saturated or unsaturated,
including epoxides and thioepoxides; A-T are independently selected
from hydrogen, R.sub.1, R.sub.2, R.sub.3, F, Cl, Br, I, CN,
OR.sub.1, SR.sub.1, NR.sub.1R.sub.2, N(.dbd.O).sub.2,
NR.sub.1OR.sub.2, ONR.sub.1R.sub.2, SOR.sub.1, SO.sub.2R.sub.1,
SO.sub.3R.sub.1, SONR.sub.1R.sub.2, SO.sub.2NR.sub.1R.sub.2,
SO.sub.3NR.sub.1R.sub.2, P(R.sub.1).sub.3,
P(.dbd.O)(R.sub.1).sub.3, Si(R.sub.1).sub.3, B(R.sub.1).sub.2,
(C.dbd.X)R.sub.3 or X(C.dbd.X)R.sub.3 where X is selected from
sulfur, oxygen and nitrogen; R.sub.1 and R.sub.2 are each
independently selected from C.sub.1-C.sub.20 alkyl (branched and/or
straight chained), C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and/or straight chained), C.sub.2-C.sub.10 alkynyl (branched and/or
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.1, SR.sub.1, NR.sub.1R.sub.2, N(.dbd.O).sub.2,
NR.sub.1OR.sub.2, ONR.sub.1R.sub.2, SOR.sub.1, SO.sub.2R.sub.1,
SO.sub.3R.sub.1, SONR.sub.1R.sub.2, SO.sub.2NR.sub.1R.sub.2,
SO.sub.3NR.sub.1R.sub.2, P(R.sub.1).sub.3,
P(.dbd.O)(R.sub.1).sub.3, Si(R.sub.1).sub.3,
B(R.sub.1).sub.2]alkyl; R.sub.3 is selected from R.sub.1, R.sub.2,
CN, COR.sub.1, CO.sub.2R.sub.1, OR.sub.1, SR.sub.1,
NR.sub.1R.sub.2, N(.dbd.O).sub.2, NR.sub.1OR.sub.2,
ONR.sub.1R.sub.2, SOR.sub.1, SO.sub.2R.sub.1, SO.sub.3R.sub.1,
SONR.sub.1R.sub.2, SO.sub.2NR.sub.1R.sub.2,
SO.sub.3NR.sub.1R.sub.2, P(R.sub.1).sub.3,
P(.dbd.O)(R.sub.1).sub.3, Si(R.sub.1).sub.3, B(R.sub.1).sub.2; A
connected to B (or C), D (or E, R (or Q), P (or O) or S (or T) is a
selection of C.sub.1-C.sub.8 disubstituted (fused) saturated or
unsaturated carbocyclic or heterocyclic rings further substituted
by R.sub.3, (C.dbd.X)R.sub.3 and X(C.dbd.X)R.sub.3, including
epoxides and thioepoxides; J connected to I (or H), G (or F), K (or
L), M (or N) or S (or T) is a selection of C.sub.1-C.sub.8
disubstituted (fused) saturated and unsaturated carbocyclic or
heterocyclic rings further substituted by R.sub.3, (C.dbd.X)R.sub.3
and X(C.dbd.X)R.sub.3, including epoxides and thioepoxides; D (or
E) connected to B (or C) or G (or F); I (or H) connected to G (or
F); P (or O) connected to R (or Q) or M (or N); K (or L) connected
to N (or M) is a selection of C.sub.1-C.sub.8 disubstituted (fused)
saturated or unsaturated carbocyclic or heterocyclic rings
substituted by R.sub.3, (C.dbd.X)R.sub.3 and X(C.dbd.X)R.sub.3,
including epoxides and thioepoxides; B and C, D and E, R and Q, P
and O, I and H, G and F, K and L, M and N or S and T are .dbd.X
where X is selected from sulfur, oxygen, nitrogen, NR.sub.1R.sub.2,
and .dbd.CR.sub.1R.sub.2 26wherein: n is 0-10 atoms selected from
carbon, oxygen, nitrogen, sulfur, phosphorus, silicon, boron,
arsenic and selenium, wherein the ring defined by said atoms is
saturated or unsaturated, including epoxides and thioepoxides;
A'-T' are independently selected from hydrogen, R.sub.4, R.sub.5,
R.sub.6, F, Cl, Br, I, CN, COR.sub.4, CO.sub.2R.sub.4, OR.sub.4,
SR.sub.4, NR.sub.4R.sub.5, CONR.sub.4R.sub.5, N(.dbd.O).sub.2,
NR.sub.4OR.sub.5, ONR.sub.4R.sub.5, SOR.sub.4, SO.sub.2R.sub.4,
SO.sub.3R.sub.4, SONR.sub.4R.sub.5, S0.sub.2NR4R.sub.5,
S0.sub.3NR.sub.4R.sub.5, P(R.sub.4).sub.3,
P(.dbd.O)(R.sub.4).sub.3, Si(R.sub.4).sub.3, B(R.sub.4).sub.2,
(C.dbd.X)R.sub.6 or X(C.dbd.X)R.sub.6 where X is selected from
sulfur, oxygen and nitrogen; R.sub.4 and R.sub.5 are each
independently selected from C.sub.1-C.sub.20 alkyl (branched and/or
straight chained), C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and/or straight chained), C.sub.2-C.sub.10 alkynyl (branched and/or
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.4, SR.sub.4, NR.sub.4R.sub.5, N(.dbd.O).sub.2,
NR.sub.4OR.sub.5, ONR.sub.4R.sub.5, SOR.sub.4, SO.sub.2R.sub.4,
SO.sub.3R.sub.4, SONR.sub.4R.sub.5, SO.sub.2NR.sub.4R.sub.5,
SO.sub.3NR.sub.4R.sub.5, P(R.sub.4).sub.3,
P(.dbd.O)(R.sub.4).sub.3, Si(R.sub.4).sub.3,
B(P.sub.4).sub.2]alkyl; R.sub.6 is selected from R.sub.4, R.sub.5,
CN, COR.sub.4, CO.sub.2R.sub.4, OR.sub.4, SR.sub.4,
NR.sub.4R.sub.5, N(.dbd.O).sub.2, NR.sub.4OR.sub.5,
ONR.sub.4R.sub.5, SOR.sub.4, SO.sub.2R.sub.4, SO.sub.3R.sub.4,
SONR.sub.4R.sub.5, SO.sub.2NR.sub.4R.sub.5,
SO.sub.3NR.sub.4R.sub.5, P(R.sub.4).sub.3,
P(.dbd.O)(R.sub.4).sub.3, Si(R.sub.4).sub.3, B(R.sub.4).sub.2; E'
and R' or H' and O' is a C.sub.2-C.sub.8 saturated or unsaturated
carbocyclic or heterocyclic ring system further substituted by
R.sub.6, including epoxides and thioepoxides; O' connected to M'
(or N') or Q' (or P'); R' connected to Q' (or P') or S' (or T'); S'
(or T') connected to A' (or B'); A' (or B') connected to C' (or
D'); E' connected to C' (or D') or F' (or G'); H' connected to I';
I' connected to J'; J' connected to K'; K' connected to L'; L'
connected to M' (or N') are C.sub.1-C.sub.8 disubstituted (fused)
saturated or unsaturated carbocyclic or heterocyclic ring systems
further substituted by R.sub.6, (C.dbd.X)R.sub.6 and
X(C.dbd.X)R.sub.6, including epoxides and thioepoxides; A', B' and
C', D' and F', G' and M', N' and P', Q' and S', T' are .dbd.X where
X is selected from sulfur, oxygen, nitrogen, NR.sub.4R.sub.5,
(C.dbd.X)R.sub.6, X(C.dbd.X)R.sub.6, and .dbd.CR.sub.7R.sub.8;
R.sub.7 and R.sub.8 are each independently selected from R.sub.6,
(C.dbd.X)R.sub.6 and X(C.dbd.X)R.sub.6 27wherein: n is 0-10 atoms
selected from carbon, oxygen, nitrogen, sulfur, phosphorus,
silicon, boron, arsenic and selenium, wherein the ring defined by
said atoms is saturated or unsaturated, including epoxides and
thioepoxides; A.sup.1-T.sup.1 are independently selected from
hydrogen, R.sub.9, R.sub.10, R.sub.11, F, Cl, Br, I, CN, OR.sub.9,
SR.sub.9, NR.sub.9R.sub.10, N(.dbd.O).sub.2, NR.sub.9OR.sub.10,
ONR.sub.9R.sub.10, SOR.sub.9, SO.sub.2R.sub.9, SO.sub.3R.sub.9,
SONR.sub.9R.sub.10, SO.sub.2NR.sub.9R.sub.10,
SO.sub.3NR.sub.9R.sub.10, P(R.sub.9).sub.3,
P(.dbd.O)(R.sub.9).sub.3, Si(R.sub.9).sub.3, B(R.sub.9).sub.2,
(C.dbd.X)R.sub.11 or X(C.dbd.X)R.sub.11 where X is selected from
sulfur, oxygen and nitrogen; R.sub.9 and R.sub.10 are each
independently selected from C.sub.1-C.sub.20 alkyl (branched and
straight chained), C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and straight chained), C.sub.2-C.sub.10 alkynyl (branched and
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.9, SR.sub.9, NR.sub.9R.sub.10, N(.dbd.O).sub.2,
NR.sub.9OR.sub.10, ONR.sub.9R.sub.10, SOR.sub.9, SO.sub.2R.sub.9,
SO.sub.3R.sub.9, SONR.sub.9R.sub.10, SO.sub.2NR.sub.9R.sub.10,
SO.sub.3NR.sub.9R.sub.10, P(R.sub.9).sub.3,
P(.dbd.O)(R.sub.9).sub.3, Si(R.sub.9).sub.3,
B(R.sub.9).sub.2]alkyl; R.sub.11 is selected from R.sub.9,
R.sub.10, CN, COR.sub.9, CO.sub.2R.sub.9, OR.sub.9, SR.sub.9,
NR.sub.9R.sub.10, N(.dbd.O).sub.2, NR.sub.9OR.sub.10,
ONR.sub.9R.sub.10, SOR.sub.9, SO.sub.2R.sub.9, SO.sub.3R.sub.9,
SONR.sub.9R.sub.10, SO.sub.2NR.sub.9R.sub.10,
SO.sub.3NR.sub.9R.sub.10, P(R.sub.9).sub.3,
P(.dbd.O)(R.sub.9).sub.3, Si(R.sub.9).sub.3, B(R.sub.9).sub.2;
B.sup.1 and R.sup.1, E.sup.1 and .sup.1 and .sup.1 and M.sup.1 are
selected from a C.sub.2-C.sub.8 saturated or unsaturated
carbocyclic or heterocyclic ring system further substituted by
R.sub.11, including epoxides and thioepoxides; A.sup.1 (or .sup.1
connected to .sup.1 (or .sup.1) or T.sup.1 (or S.sup.1) B.sup.1
connected to .sup.1 (or .sup.1) or C.sup.1 (or D.sup.1) E.sup.1
connected to .sup.1 or C.sup.1 (or D.sup.1); .sup.1 connected to
.sup.1 (or F.sup.1); G.sup.1 (or H.sup.1) connected to .sup.1 (or
F.sup.1) or I.sup.1 (or J.sup.1); K.sup.1 (or L.sup.1) connected to
I.sup.1 (or J.sup.1) or M.sup.1; M.sup.1 connected to O.sup.1 (or
N.sup.1); .sup.1 connected O.sup.1 (or N.sup.1) or P.sup.1 (or
Q.sup.1); R.sup.1 connected P.sup.1 (or Q.sup.1) or S.sup.1 (or
T.sup.1) are C.sub.1-C.sub.8 disubstituted (fused) saturated or
unsaturated carbocyclic or heterocyclic ring systems further
substituted by R.sub.11, (C.dbd.X)R.sub.11 and X(C.dbd.X)R.sub.11,
including epoxides and thioepoxides; A.sup.1, and , and C.sup.1,
D.sup.1 and F.sup.1, and G.sup.1, H.sup.1 and I.sup.1, J.sup.1 and
K.sup.1, L.sup.1 and N.sup.1, O.sup.1 and P.sup.1, Q.sup.1 and
S.sup.1, T.sup.1 are .dbd.X where X is selected from sulfur,
oxygen, nitrogen, NR.sub.9R.sub.10, including (C.dbd.X)R.sub.11 and
X(C.dbd.X)R.sub.11, and .dbd.CR.sub.12R.sub.13; R.sub.12 and
R.sub.13 are independently selected from R.sub.11,
(C.dbd.X)R.sub.11 and X(C.dbd.X)R.sub.11 28wherein: n is 0-10 atoms
selected from carbon, oxygen, nitrogen, sulfur, phosphorus,
silicon, boron, arsenic and selenium, wherein the ring defined by
said atoms is saturated or unsaturated, including epoxides and
thioepoxides; A.sup.2-X.sup.2 are independently selected from
hydrogen, R.sub.14, R.sub.15, R.sub.16, F, Cl, Br, I, CN,
OR.sub.14, SR.sub.14, NR.sub.14R.sub.15, N(.dbd.O).sub.2,
NR.sub.14OR.sub.15, ONR.sub.14R.sub.15, SOR.sub.14,
SO.sub.2R.sub.14, SO.sub.3R.sub.14, SONR.sub.14R.sub.15,
SO.sub.2NR.sub.14R.sub.15, SO.sub.3NR.sub.14R.sub.15- ,
P(R.sub.14).sub.3, P(.dbd.O)(R.sub.14).sub.3, Si(R.sub.14).sub.3,
B(R.sub.14), (C.dbd.Y)R.sub.16 or Y(C.dbd.Y)R.sub.16 where Y is
selected from sulfur, oxygen and nitrogen; R.sub.14 and R.sub.15
are each independently selected from C.sub.1-C.sub.20 alkyl
(branched and/or straight chained), C.sub.1-C.sub.20 arylalkyl,
C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14
heteroaryl, C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl
(branched and/or straight chained), C.sub.2-C.sub.10 alkynyl
(branched and/or straight chained), C.sub.1-C.sub.10
heteroarylalkyl, C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10
haloalkyl, dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10
[CN, OR.sub.14, SR.sub.14, NR.sub.14R.sub.10, N(.dbd.O).sub.2,
NR.sub.14OR.sub.15, ONR.sub.14R.sub.15, SOR.sub.14,
SO.sub.2R.sub.14, SO.sub.3R.sub.14, SONR.sub.14R.sub.15,
SO.sub.2NR.sub.14R.sub.15, SO.sub.3NR.sub.14R.sub.15,
P(R.sub.14).sub.3, P(.dbd.O)(R.sub.14).sub.3, Si(R.sub.14).sub.3,
B(R.sub.14).sub.2]alkyl; R.sub.16 is selected from R.sub.14,
R.sub.15, CN, COR.sub.14, CO.sub.2R.sub.15, OR.sub.14, SR.sub.14,
NR.sub.14R.sub.15, N(.dbd.O).sub.2, NR.sub.14OR.sub.15,
ONR.sub.14R.sub.15, SOR.sub.14, SO.sub.2R.sub.14, SO.sub.3R.sub.14,
SONR.sub.14R.sub.15, SO.sub.2NR.sub.14R.sub.15,
SO.sub.3NR.sub.14R.sub.15, P(R.sub.14).sub.3,
P(.dbd.O)(R.sub.14).sub.3, Si(R.sub.14).sub.3, B(R.sub.14).sub.2;
E.sup.2 and V.sup.2, H.sup.2 and S.sup.2, and I.sup.2 and P.sup.2
are C.sub.2-C.sub.8 saturated or unsaturated carbocyclic or
heterocyclic ring system further substituted by R.sub.16, including
epoxides and thioepoxides; A.sup.2 (or B.sup.2) connected to
C.sup.2 (or D.sup.2) or W.sup.2 (or X.sup.2); E.sup.2 connected to
C.sup.2 (or D.sup.2) or F.sup.2 (or G.sup.2); H.sup.2 connected to
F.sup.2 (or G.sup.2) or I.sup.2; I.sup.2 connected to J.sup.2 (or
K.sup.2); L.sup.2 (or M.sup.2) connected to J.sup.2 (or K.sup.2) or
N.sup.2 (or O.sup.2); R.sup.2 (or Q.sup.2) connected to P.sup.2 or
S.sup.2; V.sup.2 connected to U.sup.2 (or T.sup.2) or W.sup.2 (or
X.sup.2) are C.sub.1-C.sub.8 disubstituted (fused) saturated or
unsaturated carbocyclic or heterocyclic ring systems further
substituted by R.sub.16, (C.dbd.Y)R.sub.16 and Y(C.dbd.Y)R.sub.16,
including epoxides and thioepoxides; A.sup.2, B.sup.2; C.sup.2,
D.sup.2; F.sup.2, G.sup.2; J.sup.2, K.sup.2; L.sup.2, M.sup.2;
N.sup.2, O.sup.2; Q.sup.2, R.sup.2; U.sup.2, T.sup.2 and X.sup.2,
W.sup.2 are .dbd.Y where Y is selected from sulfur, oxygen,
nitrogen, NR.sub.14R.sub.15 and .dbd.CR.sub.17R.sub.18; R.sub.17
and R.sub.18 are independently selected from R.sub.16,
(C.dbd.Y)R.sub.16 and Y(C.dbd.Y)R.sub.16 29wherein: n is 0-10 atoms
selected from carbon, oxygen, nitrogen, sulfur, phosphorus,
silicon, boron, arsenic and selenium, wherein the ring defined by
said atoms is saturated or unsaturated, including epoxides and
thioepoxides; A.sup.3-Z.sup.3 are independently selected from
hydrogen, R.sub.19, R.sub.20, R.sub.21, F, Cl, Br, I, CN,
OR.sub.19, SR.sub.19, NR.sub.19R.sub.20, N(.dbd.O).sub.2,
NR.sub.19OR.sub.20, ONR.sub.19R.sub.20, SOR.sub.19,
SO.sub.2R.sub.19, SO.sub.3R.sub.19, SONR.sub.19R.sub.20,
SO.sub.2NR.sub.19R.sub.20, SO.sub.3NR.sub.19R.sub.20,
P(R,.sub.9).sub.3, P(O)(R.sub.19).sub.3, Si(R.sub.19).sub.3,
B(R.sub.19).sub.2, (C.dbd..O slashed.)R.sub.21 or .O
slashed.(C.dbd..O slashed.)R.sub.21 where .O slashed. is sullur,
oxygen and nitrogen; R.sub.19 and R.sub.20 are each independently
selected from C.sub.1-C.sub.20 alkyl (branched and/or straight
chained), C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8 cycloalkyl,
C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and/or straight chained), C.sub.2-C.sub.10 alkynyl (branched and/or
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.19, SR.sub.19, NR.sub.19R.sub.20, N(.dbd.O).sub.2,
NR.sub.19OR.sub.20, ONR.sub.19R.sub.20, SOR.sub.19,
SO.sub.2R.sub.19, SO.sub.3R.sub.19, SONR.sub.19R.sub.20,
SO.sub.2NR.sub.19R.sub.20, SO.sub.3NR.sub.19R.sub.20,
P(R.sub.19).sub.3, P(.dbd.O)(R.sub.19).sub.3, Si(R.sub.19).sub.3,
B(R.sub.19).sub.2]alkyl; R.sub.21 is selected from R.sub.19,
R.sub.20, CN, COR.sub.19, CO.sub.2R.sub.19, OR.sub.19, SR.sub.19,
NR.sub.19R.sub.20, N(.dbd.O).sub.2, NR.sub.19OR.sub.20,
ONR.sub.19R.sub.20, SOR.sub.19, SO.sub.2R.sub.19, SO.sub.3R.sub.19,
SONR.sub.19R.sub.20, SO.sub.2NR.sub.19R.sub.20,
SO.sub.3NR.sub.19R.sub.20, P(R.sub.19).sub.3,
P(.dbd.O)(R.sub.19).sub.3, Si(R.sub.19).sub.3, B(R.sub.19).sub.2;
D.sup.3 connected to X.sup.3 is a C.sub.2-C.sub.8 saturated or
unsaturated carbocyclic or heterocyclic ring system further
substituted by R.sub.21, including epoxides and thioepoxides;
A.sup.3 (or .sup.3) connected to B.sup.3 (or C.sup.3) or E.sup.3
(or X); D.sup.3 connected to B.sup.3 (or C.sup.3) or E.sup.3 (or
F.sup.3); G.sup.3 (or H.sup.3) connected to E.sup.3 (or F.sup.3) or
I.sup.3 (or J.sup.3); L.sup.3 (or K.sup.3) connected to I.sup.3 (or
J.sup.3) or M.sup.3 (or N.sup.3); O.sup.3 (or .sup.3) connected to
N.sup.3 (or M.sup.3) or P.sup.3 (or Q.sup.3). S.sup.3 (or R.sup.3)
connected to Q.sup.3 (or P.sup.3) or U.sup.3 (or T.sup.3). W.sup.3
(or V.sup.3) connected to U.sup.3 (or T.sup.3) or X.sup.3; X.sup.3
connected to Y.sup.3 (or Z.sup.3) are C.sub.1-C.sub.8 disubstituted
(fused) saturated or unsaturated carbocyclic or heterocyclic ring
systems further substituted by R.sub.21, (C.dbd..O
slashed.)R.sub.21 and .O slashed. (C.dbd..O slashed.)R.sub.21,
including epoxides and thioepoxides; A.sup.3, .sup.3; B.sup.3,
C.sup.3; E.sup.3, F.sup.3; G.sup.3, H.sup.3; I.sup.3, J.sup.3;
K.sup.3, L.sup.3; M.sup.3, N.sup.3; O.sup.3, .sup.3; Q.sup.3,
P.sup.3, S.sup.3, R.sup.3, U.sup.3, T.sup.3, W.sup.3, V.sup.3, and
Z.sup.3, Y.sup.3 are .dbd..O slashed. where .O slashed. is selected
from sulfur, oxygen, nitrogen, NR.sub.19R.sub.20, and
.dbd.CR.sub.22R.sub.23; and R.sub.22 and R.sub.23 are selected from
R.sub.21, (C.dbd..O slashed.)R.sub.21 and .O slashed.(C.dbd..O
slashed.)R.sub.21; and which chemical agent or derivative or
chemical analogue thereof is capable of modulating PKC activity,
PKC-gene expression or PKC enzyme turnover and wherein said
chemical agent or its derivatives or chemical analogues is
administered for a time and under conditions sufficient to
stimulate the activation or function of said promoter.
63. A method according to claim 60 wherein the chemical agent is
represented by the general formula (VI): 30wherein: R.sub.24,
R.sub.25 and R.sub.26 are independently selected from hydrogen,
R.sub.27, R.sub.28, F, Cl, Br, I, CN, OR.sub.27, SR.sub.27,
NR.sub.27R.sub.28, N(.dbd.O).sub.2, NR.sub.27OR.sub.28,
ONR.sub.27R.sub.28, SOR.sub.27, SO.sub.2R.sub.27, SO.sub.3R.sub.27,
SONR.sub.27R.sub.28, SO.sub.2NR.sub.27R.sub.28,
SO.sub.3NR.sub.27R.sub.28, P(R.sub.27).sub.3,
P(.dbd.O)(R.sub.27).sub.3, Si(R.sub.27).sub.3, B(R.sub.27).sub.2,
(C.dbd.X)R.sub.29 or X(C.dbd.X)R.sub.29 where X is selected from
sulfur, oxygen and nitrogen; R.sub.27 and R.sub.28 are each
independently selected from C.sub.1-C.sub.20 alkyl (branched and/or
straight chained), C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and/or straight chained), C.sub.2-C.sub.10 alkynyl (branched and/or
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.27, SR.sub.27, NR.sub.27R.sub.28, N(.dbd.O).sub.2,
NR.sub.27OR.sub.28, ONR.sub.27R.sub.28, SOR.sub.27,
SO.sub.2R.sub.27, SO.sub.3R.sub.27, SONR.sub.27R.sub.28,
SO.sub.2NR.sub.27R.sub.28, SO.sub.3NR.sub.27R.sub.28,
P(R.sub.27).sub.3, P(.dbd.O)(R.sub.27).sub.3, Si(R.sub.27).sub.3,
B(R.sub.27).sub.2]alkyl; R.sub.29 is selected from R.sub.27,
R.sub.28, CN, COR.sub.27, CO.sub.2R.sub.27, OR.sub.27, SR.sub.27,
NR.sub.27R.sub.28, N(.dbd.O).sub.2, NR.sub.27OR.sub.28,
ONR.sub.27R.sub.28, SOR.sub.27, S0.sub.2R.sub.27, SO.sub.3R.sub.27,
SONR.sub.27R.sub.28, SO.sub.2NR.sub.27R.sub.28,
SO.sub.3NR.sub.27R.sub.28, P(R.sub.27).sub.3,
P(.dbd.O)(R.sub.27).sub.3, Si(R.sub.27).sub.3, B(R.sub.27).sub.2. A
method according to claim 2 wherein R.sub.24 is H.
64. A method according to claim 59 or 61 or 63 wherein R.sub.24 is
OAcetyl.
65. A method according to claim 59 or 61 or 63 wherein R.sub.24 is
OH.
66. A method according to claim 59 or 61 or 63 wherein R.sub.25 and
R.sub.26 are OH.
67. A method according to claim 1 or 59 or 61 or 63 wherein the
plant is of the genus selected from Acalypha, Acidoton,
Actinostemon, Adelia, Adenocline, Adenocrepis, Adenophaedra,
Adisca, Agrostistachys, Alchornea, Alchorneopsis, Alcinaeanthus,
Alcoceria, Aleurites, Amanoa, Andrachne, Angostyles, Anisophylluin,
Antidesma, Aphora, Aporosa, Aporosella, Argythamnia, Astrococcus,
Astrogyne, Baccanrea, Baliospermum, Bernardia, Beyeriopsis,
Bischofia, Blachia, Blumeodondron, Bonania, Bradleia, Breynia,
Breyniopsis, Briedelia, Buraeavia, Caperonia, Caryodendron,
Celianella, Cephalocroton, Chaenotheca, Chaetocarpus, Chamaesyce,
Cheilosa, Chiropetalum, Choriophyllum, Cicca, Chaoxylon, Cleidon,
Cleistanthus, Cluytia, Cnesmone, Cnidoscolus, Coccoceras, Codiaeum,
Coelodiscus, Conami, Conceveiba, Conceveibastrum, Conceveibum,
Corythea, Croizatia, Croton, Crotonopsis, Crozophora, Cubanthus,
Cunuria, Dactylostemon, Dalechampia, Dendrocousinsia, Diaspersus,
Didymocistus, Dimorphocalyx, Discocarpus, Ditaxis, Dodecastingma,
Drypetes, Dysopsis, Elateriospermum, Endadenium, Endospermum,
Erismanthus, Erythrocarpus, Erythrochilus, Eumecanthus, Euphorbia,
Euphorbiodendron, Excoecaria, Flueggea, Calearia, Garcia,
Gavarretia, Gelonium, Giara, Givotia, Glochidion, Clochidionopsis,
Glycydendron, Gymnanthes, Gymnosparia, Haematospermum, Hendecandra,
Hevea, Hieronima, Hieronyma, Hippocrepandra, Homalanthus,
Hymenocardia, Janipha, Jatropha, Julocroton, Lasiocroton,
Leiocarpus, Leonardia, Lepidanthus, Leucocroton, Mabea, Macaranga,
Mallotus, Manihot, Mappa, Maprounea, Melanthesa, Mercurialis,
Mettenia, Micrandra, Microdesmis, Microelus, Microstachy,
Maocroton, Monadenium, Mozinna, Neoscortechinia, Omalanthus,
Omphalea, Ophellantha, Orbicularia, Ostodes, Oxydectes, Palenga,
Pantadenia, Paradrypeptes, Pausandra, Pedilanthus, Pera, Peridium,
Petalostigma, Phyllanthus, Picrodendro, Pierardia, Pilinophytum,
Pimeleodendron, Piranhea, Platygyna, Plukenetia, Podocalyx,
Poinsettia, Poraresia, Prosartema, Pseudanthus, Pycnocoma,
Quadrasia, Reverchonia, Richeria, Richeriella, Ricinella,
Ricinocarpus, Rottlera, Sagotia, Sanwithia, Sapium, Savia,
Sclerocroton, Sebastiana, Securinega, Senefeldera, Senefilderopsis,
Serophyton, Siphonia, Spathiostemon, Spixia, Stillingia,
Strophioblachia, Synadenium, Tetracoccus, Tetraplandra,
Tetrorchidium, Thyrsanthera, Tithymalus, Trageia, Trewia,
Trigonostemon, Tyria and Xylophylla.
68. A method according to claim 67 wherein the plant is of the
genus Euphorbia.
69. A method according to claim 68 wherein the species of Euphorbia
is selected from Euphorbia aaron-rossii, Euphorbia abbreviata,
Euphorbia acuta, Euphorbia alatocaulis, Euphorbia albicaulis,
Euphorbia algomarginata, Euphorbia aliceae, Euphorbia alta,
Euphorbia anacampseros, Euphorbia andromedae, Euphorbia angusta,
Euphorbia anthonyi, Euphorbia antiguensis, Euphorbia apocynifolia,
Euphorbia arabica, Euphorbia ariensis, Euphorbia arizonica,
Euphorbia arkansana, Euphorbia arteagae, Euphorbia arundelana,
Euphorbia astroites, Euphorbia atrococca, Euphorbia baselicis,
Euphorbia batabanensis, Euphorbia bergeri, Euphorbia bermudiana,
Euphorbia bicolor, Euphorbia biformis, Euphorbia bifurcata,
Euphorbia bilobata, Euphorbia biramensis, Euphorbia biuncialis,
Euphorbia blepharostipula, Euphorbia blodgetti, Euphorbia
boerhaavioides, Euphorbia boliviana, Euphorbia bracei, Euphorbia
brachiata, Euphorbia brachycera, Euphorbia brandegee, Euphorbia
brittonii, Euphorbia caesia, Euphorbia calcicola, Euphorbia
campestris, Euphorbia candelabrum, Euphorbia capitellata, Euphorbia
carmenensis, Euphorbia carunculata, Euphorbia cayensis, Euphorbia
celastroides, Euphorbia chalicophila, Euphorbia chamaerrhodos,
Euphorbia chamaesula, Euphorbia chiapensis, Euphorbia chiogenoides,
Euphorbia cinerascens, Euphorbia clarionensis, Euphorbia colimae,
Euphorbia colorata, Euphorbia commutata, Euphorbia consoquitlae,
Euphorbia convolvuloides, Euphorbia corallifera, Euphorbia
creberrima, Euphorbia crenulata, Euphorbia cubensis, Euphorbia
cuspidata, Euphorbia cymbiformis, Euphorbia darlingtonii, Euphorbia
defoliata, Euphorbia degeneri, Euphorbia deltoidea, Euphorbia
dentata, Euphorbia depressa Euphorbia dictyosperma, Euphorbia
dictyosperma, Euphorbia dioeca, Euphorbia discoidalis, Euphorbia
dorsiventralis, Euphorbia drumondii, Euphorbia duclouxii, Euphorbia
dussii, Euphorbia eanophylla, Euphorbia eggersii, Euphorbia
eglandulosa, Euphorbia elata, Euphorbia enalla, Euphorbia
eriogonoides, Euphorbia eriophylla, Euphorbia esculaeformis,
Euphorbia espirituensis, Euphorbia esula, Euphorbia excisa,
Euphorbia exclusa, Euphorbia exstipitata, Euphorbia exstipulata,
Euphorbia fendleri, Euphorbia filicaulis, Euphorbia filiformis,
Euphorbia florida, Euphorbia fruticulosa, Euphorbia garber,
Euphorbia gaumerii, Euphorbia gerardiana, Euphorbia geyeri,
Euphorbia glyptosperma, Euphorbia gorgonis, Euphorbia gracilior,
Euphorbia gracillima, Euphorbia gradyi, Euphorbia graminea,
Euphorbia graminiea Euphorbia grisea, Euphorbia guadalajarana,
Euphorbia guanarensis, Euphorbia gymnadenia, Euphorbia haematantha,
Euphorbia hedyotoides, Euphorbia heldrichii, Euphorbia helenae,
Euphorbia helleri, Euphorbia helwigii, Euphorbia henricksonii,
Euphorbia heterophylla, Euphorbia hexagona, Euphorbia hexagonoides,
Euphorbia hinkleyorum, Euphorbia hintonii, Euphorbia hirtula,
Euphorbia hirta, Euphorbia hooveri, Euphorbia humistrata, Euphorbia
hypericifolia, Euphorbia inundata, Euphorbia involuta, Euphorbia
jaliscensis, Euphorbia jejuna, Euphorbia johnston, Euphorbia
juttae, Euphorbia knuthii, Euphorbia lasiocarpa, Euphorbia lata,
Euphorbia latazi, Euphorbia latericolor, Euphorbia laxiflora
Euphorbia lecheoides, Euphorbia ledienii, Euphorbia leucophylla,
Euphorbia lineata, Euphorbia linguiformis, Euphorbia longecornuta,
Euphorbia longepetiolata, Euphorbia longeramosa, Euphorbia
longinsulicola, Euphorbia longipila, Euphorbia lupulina, Euphorbia
lurida, Euphorbia lycioides, Euphorbia macropodoides, macvaughiana,
Euphorbia manca, Euphorbia mandoniana, Euphorbia mangleti,
Euphorbia mango, Euphorbia marylandica, Euphorbia mayana, Euphorbia
melanadenia, Euphorbia melanocarpa, Euphorbia meridensis, Euphorbia
mertonii, Euphorbia mexiae, Euphorbia microcephala, Euphorbia
microclada, Euphorbia micromera, Euphorbia misella, Euphorbia
missurica, Euphorbia montana, Euphorbia montereyana, Euphorbia
multicaulis, Euphorbia multiformis, Euphorbia multinodis, Euphorbia
multiseta, Euphorbia muscicola, Euphorbia neomexicana, Euphorbia
nephradenia, Euphorbia niqueroana, Euphorbia oaxacana, Euphorbia
occidentalis, Euphorbia odontodenia, Euiphorbia olivacea, Euphorbia
olowaluana, Euphorbia opthalmica, Euphorbia ovata, Euphorbia
pachypoda, Euphorbia pachyrhiza, Euphorbia padifolia, Euphorbia
palmeri, Euphorbia paludicola, Euphorbia parciflora, Euphorbia
parishii, Euphorbia parryi, Euphorbia paxiana, Euphorbia
pediculifera, Euphorbia peplidion, Euphorbia peploides, Euphorbia
peplus, Euphorbia pergamena, Euphorbia perlignea, Euphorbia
petaloidea, Euphorbia petaloidea, Euphorbia petrina, Euphorbia
picachensis, Euphorbia pilosula, Euphorbia pilulifera, Euphorbia
pinariona, Euphorbia pinetorum, Euphorbia pionosperma, Euphorbia
platysperma, Euphorbia plicata, Euphorbia poeppigii, Euphorbia
poliosperma, Euphorbia polycarpa, Euphorbia polycnemoides,
Euphorbia polyphylla, Euphorbia portoricensis, Euphorbia
portulacoides Euphorbia portulana, Euphorbia preslii, Euphorbia
prostrata, Euphorbia pteroneura, Euphorbia pycnanthema, Euphorbia
ramosa, Euphorbia rapulum, Euphorbia remyi, Euphorbia retroscabra,
Euphorbia revoluta, Euphorbia rivularis, Euphorbia robusta,
Euphorbia romosa, Eiphorbia rubida, Euphorbia rubrosperma,
Euphorbia rupicola, Euphorbia sanmartensis, Euphorbia saxatilis M.
Bieb, Euphorbia schizoloba, Euphorbia sclerocyathium, Euphorbia
scopulorum, Euphorbia senilis, Euphorbia serpyllifolia, Euphorbia
serrula, Euphorbia setiloba Engelm, Euphorbia sonorae, Euphorbia
soobyi, Euphorbia sparsiflora, Euphorbia sphaerosperna, Euphorbia
syphilitica, Euphorbia spruceana, Euphorbia subcoerulea, Euphorbia
stellata, Euphorbia submammilaris, Euphorbia subpeltata, Euphorbia
subpubens, Euphorbia subreniforme, Euphorbia subtrifoliata,
Euphorbia succedanea, Euphorbia tamaulipasana, Euphorbia
telephioides, Euphorbia tenuissima, Euphorbia tetrapora, Euphorbia
tirucalli, Euphorbia tomentella, Euphorbia tomentosa, Euphorbia
torralbasii, Euphorbia tovariensis, Euphorbia trachysperma,
Euphorbia tricolor, Euphorbia troyana, Euphorbia tuerckheimii,
Euphorbia turczaninowii, Euphorbia unibellulata, Euphorbia
undulata, Euphorbia vermiformis, Euphorbia versicolor, Euphorbia
villifera, Euphorbia violacea, Euphorbia whitei, Euphorbia xanti
Engelm, Euphorbia xylopoda Greenm., Euiphorbia yayalesia Urb.,
Euphorbia yungasensis, Euphorbia zeravschanica and Euphorbia
zinniiflora.
70. A method according to claim 69 wherein the species of Euphorbia
is Euphorbia peplus.
71. A method according to claim 59 or 61 or 63 wherein the chemical
agent is a jatrophane or a derivative thereof or a pharmaceutically
acceptable salt of these.
72. A method according to claim 71 wherein said derivative is an
ester derivative.
73. A method according to claim 71 wherein said derivative is an
acetylated derivative.
74. A method according to claim 59 or 61 or 63 wherein said
chemical agent is a peptuane or a derivative thereof or a
pharmaceutically acceptable salt of these.
75. A method according to claim 74 wherein said derivative is an
ester derivative.
76. A method according to claim 74 wherein said derivative is an
acetylated derivative.
77. A method according to claim 59 or 61 or 63 wherein said
chemical agent is a paraliane or a derivative thereof or a
pharmaceutically acceptable salt of these.
78. A method according to claim 77 wherein said derivative is an
ester derivative.
79. A method according to claim 77 wherein said derivative is an
acetylated derivative.
80. A method according to claim 59 or 61 or 63 wherein said
compound is an angeloyl-substituted ingenane or a derivative
thereof or a pharmaceutically acceptable salt of these.
81. A method according to claim 80 wherein said derivative is an
acetylated derivative.
82. A method according to claim 80 wherein said jatrophane is of
conformation 2.
83. A method according to claim 71 or 74 or 77 or 80 wherein the
derivative comprises a substitution as represented in any one of
general formulae (I)-(VI).
84. A method according to claim 59 or 61 or 63 wherein said
compound is
5,8,9,10,14-pentaacetoxy-3-benzoyloxy-15-hydroxypepluane (pepluane)
or a derivative thereof or a pharmaceutically acceptable salt of
these.
85. A method according to claim 84 wherein said derivative is an
ester derivative.
86. A method according to claim 59 or 61 or 63 wherein said
compound is
2,3,5,7,15-pentaacetoxy-9-nicotinoyloxy-14-oxojatropha-6(17),
11E-diene (jatrophane 1) or a derivative thereof or a
pharmaceutically acceptable salt of these.
87. A method according to claim 86 wherein said derivative is an
ester derivative.
88. A method according to claim 59 or 61 or 63 wherein said
compound is 2,5,7,8,9,14-hexaacetoxy-3-benzoyloxy-15-hydroxy
jatropha-6(17), 11E-diene (jatrophane 2) or a derivative thereof or
a pharmaceutically acceptable salt of these.
89. A method according to claim 88 wherein said derivative is an
ester derivative.
90. A method according to claim 59 or 61 or 63 wherein said
compound is
2,5,14-triacetoxy-3-benzoyloxy-8,15-dihydroxy-7-isobutyroyloxy-9-nicotino-
yloxyjatropha-6(17), 11E-diene (jatrophane 3) or a derivative
thereof or a pharmaceutically acceptable salt of these.
91. A method according to claim 90 wherein said derivative is an
ester derivative.
92. A method according to claim 59 or 61 or 63 wherein said
compound is
2,5,9,14-tetraacetoxy-3-benzoyloxy-8,15-dihydroxy-7-isobutyroyloxyjatroph-
a-6(17),11E-diene) (jatrophane 4) or a derivative thereof or a
pharmaceutically acceptable salt of these.
93. A method according to claim 92 wherein said derivative is an
ester derivative.
94. A method according to claim 59 or 61 or 63 wherein said
compound is
2,5,7,14-tetraacetoxy-3-benzoyloxy-8,15-dihydroxy-9-nicotinoyloxyjatropha-
-6(17),11E-diene (jatrophane 5) or a derivative thereof or a
pharmaceutically acceptable salt of these.
95. A method according to claim 94 wherein said derivative is an
ester derivative.
96. A method according to claim 59 or 61 or 63 wherein said
compound is
2,5,7,9,14-pentaacetoxy-3-benzoyloxy-8,15-dihydroxyjatropha-6(17),
11E-diene (jatrophane 6) or a derivative thereof or a
pharmaceutically acceptable salt of these.
97. A method according to claim 96 wherein said derivative is an
ester derivative.
98. A method according to claim 59 or 61 or 63 wherein said
compound is 20-O-acetyl-ingenol-3-angelate or a derivative thereof
or a pharmaceutically acceptable salt of these.
99. A method according to claim 98 wherein said derivative is an
ester derivative.
100. A method according to claim 71 or 74 or 77 or 80 or 84 or 86
or 88 or 90 or 92 or 94 or 96 or 98 wherein said compound is
provided in the form of a composition comprising a
pharmaceutically- or cosmetically-acceptable carrier.
101. A method according to claim 100 wherein said carrier is
selected from .beta.-alanine betaine hydrochloride and
t-4-hydroxy-N,N-dimethylproline.
102. A method for the treatment or prophylaxis of a PKC-related
condition in a subject, said method comprising the administration
to said subject of a symptom-ameliorating or immunopotentiating
effective amount of a macrocyclic diterpene obtainable from a
Euphorbiaceae plant or its botanical or horticultural relative,
said macrocyclic diterpene being selected from an ingenane,
pepluane or jatrophane, or a derivative or chemical analogue
thereof, having the structure represented by any one of the general
formulae (I)-(V) 31wherein: n is 0-10 atoms selected from carbon,
oxygen, nitrogen, sulfur, phosphorus, silicon, boron, arsenic and
selenium, wherein the ring defined by said atoms is saturated or
unsaturated, including epoxides and thioepoxides; A-T are
independently selected from hydrogen, R.sub.1, R.sub.2, R.sub.3, F,
Cl, Br, I, CN, OR.sub.1, SR.sub.1, NR.sub.1R.sub.2,
N(.dbd.O).sub.2, NR.sub.1OR.sub.2, ONR.sub.1R.sub.2, SOR.sub.1,
SO.sub.2R.sub.1, SO.sub.3R.sub.1, SONR.sub.1R.sub.2,
SO.sub.2NR.sub.1R.sub.2, SO.sub.3NR.sub.1R.sub.2, P(R.sub.1).sub.3,
P(.dbd.O)(R.sub.1).sub.3, Si(R.sub.1).sub.3, B(R.sub.1).sub.2,
(C.dbd.X)R.sub.3 or X(C.dbd.X)R.sub.3 where X is selected from
sulfur, oxygen and nitrogen; R.sub.1 and R.sub.2 are each
independently selected from C.sub.1-C.sub.20 alkyl (branched and/or
straight chained), C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and/or straight chained), C.sub.2-C.sub.10 alkynyl (branched and/or
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.1, SR.sub.1, NR.sub.1R.sub.2, N(.dbd.O).sub.2,
NR.sub.1OR.sub.2, ONR.sub.1R.sub.2, SOR.sub.1, SO.sub.2R.sub.1,
SO.sub.3R.sub.1, SONR.sub.1R.sub.2, SO.sub.2NR.sub.1R.sub.2,
SO.sub.3NR.sub.1R.sub.2, P(R.sub.1).sub.3,
P(.dbd.O)(R.sub.1).sub.3, Si(R.sub.1).sub.3,
B(R.sub.1).sub.2]alkyl; R.sub.3 is selected from R.sub.1, R.sub.2,
CN, COR.sub.1, CO.sub.2R.sub.1, OR.sub.1, SR.sub.1,
NR.sub.1R.sub.2, N(.dbd.O).sub.2, NR.sub.1OR.sub.2,
ONR.sub.1R.sub.2, SOR.sub.1, SO.sub.2R.sub.1, SO.sub.3R.sub.1,
SONR.sub.1R.sub.2, SO.sub.2NR.sub.1R.sub.2,
SO.sub.3NR.sub.1R.sub.2, P(R.sub.1).sub.3,
P(.dbd.O)(R.sub.1).sub.3, Si(R.sub.1).sub.3, B(R.sub.1).sub.2; A
connected to B (or C), D (or E, R (or Q), P (or O) or S (or T) is a
selection of C.sub.1-C.sub.8 disubstituted (fused) saturated or
unsaturated carbocyclic or heterocyclic rings further substituted
by R.sub.3, (C.dbd.X)R.sub.3 and X(C.dbd.X)R.sub.3, including
epoxides and thioepoxides; J connected to I (or H), G (or F), K (or
L), M (or N) or S (or T) is a selection of C.sub.1-C.sub.8
disubstituted (fused) saturated and unsaturated carbocyclic or
heterocyclic rings further substituted by R.sub.3, (C.dbd.X)R.sub.3
and X(C.dbd.X)R.sub.3, including epoxides and thioepoxides; D (or
E) connected to B (or C) or G (or F); I (or H) connected to G (or
F); P (or O) connected to R (or Q) or M (or N); K (or L) connected
to N (or M) is a selection of C.sub.1-C.sub.8 disubstituted (fused)
saturated or unsaturated carbocyclic or heterocyclic rings
substituted by R.sub.3, (C.dbd.X)R.sub.3 and X(C.dbd.X)R.sub.3,
including epoxides and thioepoxides; B and C, D and E, R and Q, P
and O, I and H, G and F, K and L, M and N or S and T are .dbd.X
where X is selected from sulfur, oxygen, nitrogen, NR.sub.1R.sub.2,
and .dbd.CR.sub.1R.sub.2 32wherein: n is 0-10 atoms selected from
carbon, oxygen, nitrogen, sulfur, phosphorus, silicon, boron,
arsenic and selenium, wherein the ring defined by said atoms is
saturated or unsaturated, including epoxides and thioepoxides;
A'-T' are independently selected from hydrogen, R.sub.4, R.sub.5,
R.sub.6, F, Cl, Br, I, CN, COR.sub.4, CO.sub.2R.sub.4, OR.sub.4,
SR.sub.4, NR.sub.4R.sub.5, CONR.sub.4R.sub.5, N(.dbd.O).sub.2,
NR.sub.4OR.sub.5, ONR.sub.4R.sub.5, SOR.sub.4, SO.sub.2R.sub.4,
SO.sub.3R.sub.4, SONR.sub.4R.sub.5, S0.sub.2NR4R.sub.5,
S0.sub.3NR.sub.4R.sub.5, P(R.sub.4).sub.3,
P(.dbd.O)(R.sub.4).sub.3, Si(R.sub.4).sub.3, B(R.sub.4).sub.2,
(C.dbd.X)R.sub.6 or X(C.dbd.X)R.sub.6 where X is selected from
sulfur, oxygen and nitrogen; R.sub.4 and R.sub.5 are each
independently selected from C.sub.1-C.sub.20 alkyl (branched and/or
straight chained), C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and/or straight chained), C.sub.2-C.sub.10 alkynyl (branched and/or
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.4, SR.sub.4, NR.sub.4R.sub.5, N(.dbd.O).sub.2,
NR.sub.4OR.sub.5, ONR.sub.4R.sub.5, SOR.sub.4, SO.sub.2R.sub.4,
SO.sub.3R.sub.4, SONR.sub.4R.sub.5, SO.sub.2NR.sub.4R.sub.5,
SO.sub.3NR.sub.4R.sub.5, P(R.sub.4).sub.3,
P(.dbd.O)(R.sub.4).sub.3, Si(R.sub.4).sub.3,
B(P.sub.4).sub.2]alkyl; R.sub.6 is selected from R.sub.4, R.sub.5,
CN, COR.sub.4, CO.sub.2R.sub.4, OR.sub.4, SR.sub.4,
NR.sub.4R.sub.5, N(.dbd.O).sub.2, NR.sub.4OR.sub.5,
ONR.sub.4R.sub.5, SOR.sub.4, SO.sub.2R.sub.4, SO.sub.3R.sub.4,
SONR.sub.4R.sub.5, SO.sub.2NR.sub.4R.sub.5,
SO.sub.3NR.sub.4R.sub.5, P(R.sub.4).sub.3,
P(.dbd.O)(R.sub.4).sub.3, Si(R.sub.4).sub.3, B(R.sub.4).sub.2; E'
and R' or H' and O' is a C.sub.2-C.sub.8 saturated or unsaturated
carbocyclic or heterocyclic ring system further substituted by
R.sub.6, including epoxides and thioepoxides; O' connected to M'
(or N') or Q' (or P'); R' connected to Q' (or P') or S' (or T'); S'
(or T') connected to A' (or B'); A' (or B') connected to C' (or
D'); E' connected to C' (or D') or F' (or G'); H' connected to I';
I' connected to J'; J' connected to K'; K' connected to L'; L'
connected to M' (or N') are C.sub.1-C.sub.8 disubstituted (fused)
saturated or unsaturated carbocyclic or heterocyclic ring systems
further substituted by R.sub.6, (C.dbd.X)R.sub.6 and
X(C.dbd.X)R.sub.6, including epoxides and thioepoxides; A', B' and
C', D' and F', G' and M', N' and P', Q' and S', T' are .dbd.X where
X is selected from sulfur, oxygen, nitrogen, NR.sub.4R.sub.5,
(C.dbd.X)R.sub.6, X(C.dbd.X)R.sub.6, and .dbd.CR.sub.7R.sub.8;
R.sub.7 and R.sub.8 are each independently selected from R.sub.6,
(C.dbd.X)R.sub.6 and X(C.dbd.X)R.sub.6 33wherein: n is 0-10 atoms
selected from carbon, oxygen, nitrogen, sulfur, phosphorus,
silicon, boron, arsenic and selenium, wherein the ring defined by
said atoms is saturated or unsaturated, including epoxides and
thioepoxides; A.sup.1-T.sup.1 are independently selected from
hydrogen, R.sub.9, R.sub.10, R.sub.11, F, Cl, Br, I, CN, OR.sub.9,
SR.sub.9, NR.sub.9R.sub.10, N(.dbd.O).sub.2, NR.sub.9OR.sub.10,
ONR.sub.9R.sub.10, SOR.sub.9, SO.sub.2R.sub.9, SO.sub.3R.sub.9,
SONR.sub.9R.sub.10, SO.sub.2NR.sub.9R.sub.10,
SO.sub.3NR.sub.9R.sub.10, P(R.sub.9).sub.3,
P(.dbd.O)(R.sub.9).sub.3, Si(R.sub.9).sub.3, B(R.sub.9).sub.2,
(C.dbd.X)R.sub.11 or X(C.dbd.X)R.sub.11 where X is selected from
sulfur, oxygen and nitrogen; R.sub.9 and R.sub.10 are each
independently selected from C.sub.1-C.sub.20 alkyl (branched and
straight chained), C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and straight chained), C.sub.2-C.sub.10 alkynyl (branched and
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.9, SR.sub.9, NR.sub.9R.sub.10, N(.dbd.O).sub.2,
NR.sub.9OR.sub.10, ONR.sub.9R.sub.10, SOR.sub.9, SO.sub.2R.sub.9,
SO.sub.3R.sub.9, SONR.sub.9R.sub.10, SO.sub.2NR.sub.9R.sub.10,
SO.sub.3NR.sub.9R.sub.10, P(R.sub.9).sub.3,
P(.dbd.O)(R.sub.9).sub.3, Si(R.sub.9).sub.3,
B(R.sub.9).sub.2]alkyl; R.sub.11 is selected from R.sub.9,
R.sub.10, CN, COR.sub.9, CO.sub.2R.sub.9, OR.sub.9, SR.sub.9,
NR.sub.9R.sub.10, N(.dbd.O).sub.2, NR.sub.9OR.sub.10,
ONR.sub.9R.sub.10, SOR.sub.9, SO.sub.2R.sub.9, SO.sub.3R.sub.9,
SONR.sub.9R.sub.10, SO.sub.2NR.sub.9R.sub.10,
SO.sub.3NR.sub.9R.sub.10, P(R.sub.9).sub.3,
P(.dbd.O)(R.sub.9).sub.3, Si(R.sub.9).sub.3, B(R.sub.9).sub.2;
B.sup.1 and R.sup.1, E.sup.1 and .sup.1 and .sup.1 and M.sup.1 are
selected from a C.sub.2-C.sub.8 saturated or unsaturated
carbocyclic or heterocyclic ring system further substituted by
R.sub.11, including epoxides and thioepoxides; A.sup.1 (or .sup.1
connected to .sup.1 (or .sup.1) or T.sup.1 (or S.sup.1) B.sup.1
connected to .sup.1 (or .sup.1) or C.sup.1 (or D.sup.1) E.sup.1
connected to .sup.1 or C.sup.1 (or D.sup.1); .sup.1 connected to
.sup.1 (or F.sup.1); G.sup.1 (or H.sup.1) connected to .sup.1 (or
F.sup.1) or I.sup.1 (or J.sup.1); K.sup.1 (or L.sup.1) connected to
I.sup.1 (or J.sup.1) or M.sup.1; M.sup.1 connected to O.sup.1 (or
N.sup.1); .sup.1 connected O.sup.1 (or N.sup.1) or P.sup.1 (or
Q.sup.1); R.sup.1 connected P.sup.1 (or Q.sup.1) or S.sup.1 (or
T.sup.1) are C.sub.1-C.sub.8 disubstituted (fused) saturated or
unsaturated carbocyclic or heterocyclic ring systems further
substituted by R.sub.11, (C.dbd.X)R.sub.11 and X(C.dbd.X)R.sub.11,
including epoxides and thioepoxides; A.sup.1, and , and C.sup.1,
D.sup.1 and F.sup.1, and G.sup.1, H.sup.1 and I.sup.1, J.sup.1 and
K.sup.1, L.sup.1 and N.sup.1, O.sup.1 and P.sup.1, Q.sup.1 and
S.sup.1, T.sup.1 are .dbd.X where X is selected from sulfur,
oxygen, nitrogen, NR.sub.9R.sub.10, including (C.dbd.X)R.sub.11 and
X(C.dbd.X)R.sub.11, and .dbd.CR.sub.12R.sub.13; R.sub.12 and
R.sub.13 are independently selected from R.sub.11,
(C.dbd.X)R.sub.11 and X(C.dbd.X)R.sub.11 34wherein: n is 0-10 atoms
selected from carbon, oxygen, nitrogen, sulfur, phosphorus,
silicon, boron, arsenic and selenium, wherein the ring defined by
said atoms is saturated or unsaturated, including epoxides and
thioepoxides; A.sup.2-X.sup.2 are independently selected from
hydrogen, R.sub.14, R.sub.15, R.sub.16, F, Cl, Br, I, CN,
OR.sub.14, SR.sub.14, NR.sub.14R.sub.15, N(.dbd.O).sub.2,
NR.sub.14OR.sub.15, ONR.sub.14R.sub.15, SOR.sub.14,
SO.sub.2R.sub.14, SO.sub.3R.sub.14, SONR.sub.14R.sub.15,
SO.sub.2NR.sub.14R.sub.15, SO.sub.3NR.sub.14R.sub.15- ,
P(R.sub.14).sub.3, P(.dbd.O)(R.sub.14).sub.3, Si(R.sub.14).sub.3,
B(R.sub.14), (C.dbd.Y)R.sub.16 or Y(C.dbd.Y)R.sub.16 where Y is
selected from sulfur, oxygen and nitrogen; R.sub.14 and R.sub.15
are each independently selected from C.sub.1-C.sub.20 alkyl
(branched and/or straight chained), C.sub.1-C.sub.20 arylalkyl,
C.sub.3-C.sub.8 cycloalkyl, C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14
heteroaryl, C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl
(branched and/or straight chained), C.sub.2-C.sub.10 alkynyl
(branched and/or straight chained), C.sub.1-C.sub.10
heteroarylalkyl, C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10
haloalkyl, dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10
[CN, OR.sub.14, SR.sub.14, NR.sub.14R.sub.10, N(.dbd.O).sub.2,
NR.sub.14OR.sub.15, ONR.sub.14R.sub.15, SOR.sub.14,
SO.sub.2R.sub.14, SO.sub.3R.sub.14, SONR.sub.14R.sub.15,
SO.sub.2NR.sub.14R.sub.15, SO.sub.3NR.sub.14R.sub.15,
P(R.sub.14).sub.3, P(.dbd.O)(R.sub.14).sub.3, Si(R.sub.14).sub.3,
B(R.sub.14).sub.2]alkyl; R.sub.16 is selected from R.sub.14,
R.sub.15, CN, COR.sub.14, CO.sub.2R.sub.15, OR.sub.14, SR.sub.14,
NR.sub.14R.sub.15, N(.dbd.O).sub.2, NR.sub.14OR.sub.15,
ONR.sub.14R.sub.15, SOR.sub.14, SO.sub.2R.sub.14, SO.sub.3R.sub.14,
SONR.sub.14R.sub.15, SO.sub.2NR.sub.14R.sub.15,
SO.sub.3NR.sub.14R.sub.15, P(R.sub.14).sub.3,
P(.dbd.O)(R.sub.14).sub.3, Si(R.sub.14).sub.3, B(R.sub.14).sub.2;
E.sup.2 and V.sup.2, H.sup.2 and S.sup.2, and I.sup.2 and P.sup.2
are C.sub.2-C.sub.8 saturated or unsaturated carbocyclic or
heterocyclic ring system further substituted by R.sub.16, including
epoxides and thioepoxides; A.sup.2 (or B.sup.2) connected to
C.sup.2 (or D.sup.2) or W.sup.2 (or X.sup.2); E.sup.2 connected to
C.sup.2 (or D.sup.2) or F.sup.2 (or G.sup.2); H.sup.2 connected to
F.sup.2 (or G.sup.2) or I.sup.2; I.sup.2 connected to J.sup.2 (or
K.sup.2); L.sup.2 (or M.sup.2) connected to J.sup.2 (or K.sup.2) or
N.sup.2 (or O.sup.2); R.sup.2 (or Q.sup.2) connected to P.sup.2 or
S.sup.2; V.sup.2 connected to U.sup.2 (or T.sup.2) or W.sup.2 (or
X.sup.2) are C.sub.1-C.sub.8 disubstituted (fused) saturated or
unsaturated carbocyclic or heterocyclic ring systems further
substituted by R.sub.16, (C.dbd.Y)R.sub.16 and Y(C.dbd.Y)R.sub.16,
including epoxides and thioepoxides; A.sup.2, B.sup.2; C.sup.2,
D.sup.2; F.sup.2, G.sup.2; J.sup.2, K.sup.2; L.sup.2, M.sup.2;
N.sup.2, O.sup.2; Q.sup.2, R.sup.2; U.sup.2, T.sup.2 and X.sup.2,
W.sup.2 are .dbd.Y where Y is selected from sulfur, oxygen,
nitrogen, NR.sub.14R.sub.15 and .dbd.CR.sub.17R.sub.18; R.sub.17
and R.sub.18 are independently selected from R.sub.16,
(C.dbd.Y)R.sub.16 and Y(C.dbd.Y)R.sub.16 35wherein: n is 0-10 atoms
selected from carbon, oxygen, nitrogen, sulfur, phosphorus,
silicon, boron, arsenic and selenium, wherein the ring defined by
said atoms is saturated or unsaturated, including epoxides and
thioepoxides; A.sup.3-Z.sup.3 are independently selected from
hydrogen, R.sub.19, R.sub.20, R.sub.21, F, Cl, Br, I, CN,
OR.sub.19, SR.sub.19, NR.sub.19R.sub.20, N(.dbd.O).sub.2,
NR.sub.19OR.sub.20, ONR.sub.19R.sub.20, SOR.sub.19,
SO.sub.2R.sub.19, SO.sub.3R.sub.19, SONR.sub.19R.sub.20,
SO.sub.2NR.sub.19R.sub.20, SO.sub.3NR.sub.19R.sub.20,
P(R,.sub.9).sub.3, P(O)(R.sub.19).sub.3, Si(R.sub.19).sub.3,
B(R.sub.19).sub.2, (C.dbd..O slashed.)R.sub.21 or .O
slashed.(C.dbd..O slashed.)R.sub.21 where .O slashed. is sullur,
oxygen and nitrogen; R.sub.19 and R.sub.20 are each independently
selected from C.sub.1-C.sub.20 alkyl (branched and/or straight
chained), C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8 cycloalkyl,
C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and/or straight chained), C.sub.2-C.sub.10 alkynyl (branched and/or
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.19, SR.sub.19, NR.sub.19R.sub.20, N(.dbd.O).sub.2,
NR.sub.19OR.sub.20, ONR.sub.19R.sub.20, SOR.sub.19,
SO.sub.2R.sub.19, SO.sub.3R.sub.19, SONR.sub.19R.sub.20,
SO.sub.2NR.sub.19R.sub.20, SO.sub.3NR.sub.19R.sub.20,
P(R.sub.19).sub.3, P(.dbd.O)(R.sub.19).sub.3, Si(R.sub.19).sub.3,
B(R.sub.19).sub.2]alkyl; R.sub.21 is selected from R.sub.19,
R.sub.20, CN, COR.sub.19, CO.sub.2R.sub.19, OR.sub.19, SR.sub.19,
NR.sub.19R.sub.20, N(.dbd.O).sub.2, NR.sub.19OR.sub.20,
ONR.sub.19R.sub.20, SOR.sub.19, SO.sub.2R.sub.19, SO.sub.3R.sub.19,
SONR.sub.19R.sub.20, SO.sub.2NR.sub.19R.sub.20,
SO.sub.3NR.sub.19R.sub.20, P(R.sub.19).sub.3,
P(.dbd.O)(R.sub.19).sub.3, Si(R.sub.19).sub.3, B(R.sub.19).sub.2;
D.sup.3 connected to X.sup.3 is a C.sub.2-C.sub.8 saturated or
unsaturated carbocyclic or heterocyclic ring system further
substituted by R.sub.21, including epoxides and thioepoxides;
A.sup.3 (or .sup.3) connected to B.sup.3 (or C.sup.3) or E.sup.3
(or X); D.sup.3 connected to B.sup.3 (or C.sup.3) or E.sup.3 (or
F.sup.3); G.sup.3 (or H.sup.3) connected to E.sup.3 (or F.sup.3) or
I.sup.3 (or J.sup.3); L.sup.3 (or K.sup.3) connected to I.sup.3 (or
J.sup.3) or M.sup.3 (or N.sup.3); O.sup.3 (or .sup.3) connected to
N.sup.3 (or M.sup.3) or P.sup.3 (or Q.sup.3). S.sup.3 (or R.sup.3)
connected to Q.sup.3 (or P.sup.3) or U.sup.3 (or T.sup.3). W.sup.3
(or V.sup.3) connected to U.sup.3 (or T.sup.3) or X.sup.3; X.sup.3
connected to Y.sup.3 (or Z.sup.3) are C.sub.1-C.sub.8 disubstituted
(fused) saturated or unsaturated carbocyclic or heterocyclic ring
systems further substituted by R.sub.21, (C.dbd..O
slashed.)R.sub.21 and .O slashed. (C.dbd..O slashed.)R.sub.21,
including epoxides and thioepoxides; A.sup.3, .sup.3; B.sup.3,
C.sup.3; E.sup.3, F.sup.3; G.sup.3, H.sup.3; I.sup.3, J.sup.3;
K.sup.3, L.sup.3; M.sup.3, N.sup.3; O.sup.3, .sup.3; Q.sup.3,
P.sup.3, S.sup.3, R.sup.3, U.sup.3, T.sup.3, W.sup.3, V.sup.3, and
Z.sup.3, Y.sup.3 are .dbd..O slashed. where .O slashed. is selected
from sulfur, oxygen, nitrogen, NR.sub.19R.sub.20, and
.dbd.CR.sub.22R.sub.23; and R.sub.22 and R.sub.23 are selected from
R.sub.21, (C.dbd..O slashed.)R.sub.21 and .O slashed.(C.dbd..O
slashed.)R.sub.21; and which chemical agent or derivative or
chemical analogue thereof is capable of modulating PKC activity,
PKC-gene expression or PKC enzyme turnover and wherein said
chemical agent or its derivatives or chemical analogues is
administered for a time and under conditions sufficient to
ameliorate one or more symptoms associated with said biological
entity and wherein said chemical agent exhibits a potency of agent
(P.sub.A) of >10, wherein the P.sub.A=.SIGMA.I.sub.V where
I.sub.V is a numerical value associated with
a particular feature as listed below:
4 Feature Value An ability to modulate PKC activity or effect +1 An
ability to induce bipolar dendritic activity +1 An ability to
displace phorbol dibutyrate from +1 binding to PKC An ability to
induce respiratory burst in leucocytes +1 An ability to stimulate
phagocytosis in peripheral +1 blood mononuclear cells Derived from
a member of the Euphorbiaceae family +1 Derived from E. peplus +3
Water extractible from the sap of Euphorbia sp. +2 An ability to
activate latent virus +4 A lower tumor promotion activity than
TPA/PMA +2
or pharmaceutically acceptable salts of these, said chemical agent
being administered for a time and under conditions sufficient to
ameliorate at least one symptom caused by or associated with said
PKC-related condition.
103. A method according to claim 102 wherein the chemical agent is
represented by the general formula (VI): 36wherein: R.sub.24,
R.sub.25 and R.sub.26 are independently selected from hydrogen,
R.sub.27, R.sub.28, F, Cl, Br, I, CN, OR.sub.27, SR.sub.27,
NR.sub.27R.sub.28, N(.dbd.O).sub.2, NR.sub.27OR.sub.28,
ONR.sub.27R.sub.28, SOR.sub.27, SO.sub.2R.sub.27, SO.sub.3R.sub.27,
SONR.sub.27R.sub.28, SO.sub.2NR.sub.27R.sub.28,
SO.sub.3NR.sub.27R.sub.28, P(R.sub.27).sub.3,
P(.dbd.O)(R.sub.27).sub.3, Si(R.sub.27).sub.3, B(R.sub.27).sub.2,
(C.dbd.X)R.sub.29 or X(C.dbd.X)R.sub.29 where X is selected from
sulfur, oxygen and nitrogen; R.sub.27 and R.sub.28 are each
independently selected from C.sub.1-C.sub.20 alkyl (branched and/or
straight chained), C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8
cycloalkyl, C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and/or straight chained), C.sub.2-C.sub.10 alkynyl (branched and/or
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.27, SR.sub.27, NR.sub.27R.sub.28, N(.dbd.O).sub.2,
NR.sub.27OR.sub.28, ONR.sub.27R.sub.28, SOR.sub.27,
SO.sub.2R.sub.27, SO.sub.3R.sub.27, SONR.sub.27R.sub.28,
SO.sub.2NR.sub.27R.sub.28, SO.sub.3NR.sub.27R.sub.28,
P(R.sub.27).sub.3, P(.dbd.O)(R.sub.27).sub.3, Si(R.sub.27).sub.3,
B(R.sub.27).sub.2]alkyl; R.sub.29 is selected from R.sub.27,
R.sub.28, CN, COR.sub.27, CO.sub.2R.sub.27, OR.sub.27, SR.sub.27,
NR.sub.27R.sub.28, N(.dbd.O).sub.2, NR.sub.27OR.sub.28,
ONR.sub.27R.sub.28, SOR.sub.27, S0.sub.2R.sub.27, SO.sub.3R.sub.27,
SONR.sub.27R.sub.28, SO.sub.2NR.sub.27R.sub.28,
SO.sub.3NR.sub.27R.sub.28, P(R.sub.27).sub.3,
P(.dbd.O)(R.sub.27).sub.3, Si(R.sub.27).sub.3,
B(R.sub.27).sub.2.
104. A method according to claim 102 wherein R.sub.24 is H.
105. A method according to claim 102 wherein R.sub.24 is
OAcetyl.
106. A method according to claim 102 wherein R.sub.24 is OH.
107. A method according to claim 102 wherein R.sub.25 and R.sub.26
are OH.
108. A method according to claim 101 wherein the plant is of the
genus selected from Acalypha, Acidoton, Actinostemon, Adelia,
Adenocline, Adenocrepis, Adenophaedra, Adisca, Agrostistachys,
Alchornea, Alchorneopsis, Alcinaeanthus, Alcoceria, Aleurites,
Amanoa, Andrachne, Angostyles, Anisophyllum, Antidesma, Aphora,
Aporosa, Aporosella, Argythamnia, Astrococcus, Astrogyne,
Baccanrea, Baliospermum, Bernardia, Beyeriopsis, Bischofia,
Blachia, Blumeodondron, Bonania, Bradleia, Breynia, Breyniopsis,
Briedelia, Buraeavia, Caperonia, Caryodendron, Celianella,
Cephalocroton, Chaenotheca, Chaetocarpus, Chamaesyce, Cheilosa,
Chiropetalum, Choriophyllum, Cicca, Chaoxylon, Cleidon,
Cleistanthus, Cluytia, Cnesmone, Cnidoscolus, Coccoceras, Codiaeum,
Coelodiscus, Conami, Conceveiba, Conceveibastrum, Coneeveibum,
Corythea, Croizatia, Croton, Crotonopsis, Crozophora, Cubanthus,
Cunuria, Dactylostemon, Dalechampia, Dendrocousinsia, Diaspersus,
Didymocistus, Dimorphocalyx, Discocarpus, Ditaxis, Dodecastingma,
Drypetes, Dysopsis, Elateriospermum, Endadenium, Endospermum,
Erismanthus, Erythrocarpus, Erythrochilis, Eumecanthus, Euphorbia,
Euphorbiodendron, Excoecaria, Flueggea, Calearia, Garcia,
Gavarretia, Gelonium, Giara, Givotia, Glochidion, Clochidionopsis,
Glycydendron, Gymnanthes, Gymnosparia, Haematospermum, Hendecandra,
Hevea, Hieronima, Hieronyma, Hippocrepandra, Homalanthus,
Hymenocardia, Janipha, Jatropha, Julocroton, Lasiocroton,
Leiocarpus, Leonardia, Lepidanthus, Leucocroton, Mabea, Macaranga,
Mallotus, Manihot, Mappa, Maprounea, Melanthesa, Mercurialis,
Mettenia, Micrandra, Microdesmis, Microelus, Microstachy,
Maocroton, Monadenium, Mozinna, Neoscortechinia, Omalanthus,
Omphalea, Ophellantha, Orbicilaria, Ostodes, Oxydectes, Palenga,
Pantadenia, Paradrypeptes, Pausandra, Pedilanthus, Pera, Peridium,
Petalostigma, Phyllanthus, Picrodendro, Pierardia, Pilinophytum,
Pimeleodendron, Piranhea, Platygyna, Plukenetia, Podocalyx,
Poinsettia, Poraresia, Prosartema, Pseudanthus, Pycnocoma,
Quadrasia, Reverchonia, Richeria, Richeriella, Ricinella,
Ricinocarpus, Rottlera, Sagotia, Sanwithia, Sapium, Savia,
Sclerocroton, Sebastiana, Securinega, Senefeldera, Senefilderopsis,
Serophyton, Siphonia, Spathiostemon, Spixia, Stillingia,
Strophioblachia, Synadenium, Tetracoccus, Tetraplandra,
Tetrorchidium, Thyrsanthera, Tithymalus, Trageia, Trewia,
Trigonostemon, Tyria and Xylophylla.
109. A method according to claim 102 wherein the plant is of the
genus Euphorbia.
110. A method according to claim 109 wherein the species of
Euphorbia is selected from Euphorbia aaron-rossii, Euphorbia
abbreviata, Euphorbia acuta, Euphorbia alatocaulis, Euphorbia
albicaulis, Euphorbia algomarginata, Euphorbia aliceae, Euphorbia
alta, Euphorbia anacampseros, Euphorbia andromedae, Euphorbia
angusta, Euphorbia anthonyi, Euphorbia antiguensis, Euphorbia
apocynifolia, Euphorbia arabica, Euphorbia ariensis, Euphorbia
arizonica, Euphorbia arkansana, Euphorbia arteagae, Euphorbia
arundelana, Euphorbia astroites, Euphorbia atrococca, Euphorbia
baselicis, Euphorbia batabanensis, Euphorbia bergeri, Euphorbia
bermudiana, Euphorbia bicolor, Euphorbia biformis, Euphorbia
bifurcata, Euphorbia bilobata, Euphorbia biramensis, Euphorbia
biuncialis, Euphorbia blepharostipula, Euphorbia blodgetti,
Etiphorbia boerhaavioides, Euphorbia boliviana, Euphorbia bracei,
Euphorbia brachiata, Euphorbia brachycera, Euphorbia brandegee,
Euphorbia brittonii, Euphorbia caesia, Euphorbia calcicola,
Euphorbia campestris, Euphorbia candelabrum, Euphorbia capitellata,
Euphorbia carmenensis, Euphorbia carunculata, Euphorbia cayensis,
Euphorbia celastroides, Euphorbia chalicophila, Euphorbia
chamaerrhodos, Euphorbia chamaesula, Euphorbia chiapensis,
Euphorbia chiogenoides, Euphorbia cinerascens, Euphorbia
clarionensis, Euphorbia colimae, Euphorbia colorata, Euphorbia
commutata, Euphorbia consoquitlae, Euphorbia convolvuloides,
Euphorbia corallifera, Euphorbia creberrima, Euphorbia crenulata,
Euphorbia cubensis, Euphorbia cuspidata, Euphorbia cymbiformis,
Euphorbia darlingtonii, Euphorbia defoliata, Euphorbia degeneri,
Euphorbia deltoidea, Euphorbia dentata, Euphorbia depressa
Euphorbia dictyosperma, Euphorbia dictyosperma, Euphorbia dioeca,
Euphorbia discoidalis, Euphorbia dorsiventralis, Euphorbia
drumondii, Euphorbia duclouxii, Euphorbia dussii, Euphorbia
eanophylla, Euphorbia eggersii, Euphorbia eglandulosa, Euphorbia
elata, Euphorbia enalla, Euphorbia eriogonoides, Euphorbia
eriophylla, Euphorbia esculaeformis, Euphorbia espirituensis,
Euphorbia esula, Euphorbia excisa, Euphorbia exclusa, Euphorbia
exstipitata, Euphorbia exstipulata, Euphorbia fendleri, Euphorbia
filicaulis, Euphorbia filiformis, Euphorbia florida, Euphorbia
fruticulosa, Euphorbia garber, Euphorbia gaumerii, Euphorbia
gerardiana, Euphorbia geyeri, Euphorbia glyptosperma, Euphorbia
gorgonis, Euphorbia gracilior, Euphorbia gracillima, Euphorbia
gradyi, Euphorbia graminea, Euphorbia graminiea Euphorbia grisea,
Euphorbia guadalajarana, Euphorbia guanarensis, Euphorbia
gymnadenia, Euphorbia haematantha, Euphorbia hedyotoides, Euphorbia
heldrichii, Euphorbia helenae, Euphorbia helleri, Euphorbia
helwigii, Euphorbia henricksonii, Euphorbia heterophylla, Euphorbia
hexagona, Euphorbia hexagonoides, Euphorbia hinkleyorum, Euphorbia
hintonii, Euphorbia hirtula, Euphorbia hirta, Euphorbia hooveri,
Euphorbia humistrata, Euphorbia hypericifolia, Euphorbia inundata,
Euphorbia involuta, Euphorbia jaliscensis, Euphorbia jejuna,
Euphorbia johnston, Euphorbia juttae, Euphorbia knuthii, Euphorbia
lasiocarpa, Euphorbia lata, Euphorbia latazi, Euphorbia
latericolor, Euphorbia laxiflora Euphorbia lecheoides, Euphorbia
ledienii, Euphorbia leucophylla, Euphorbia lineata, Euphorbia
linguiformis, Euphorbia longecornuta, Euphorbia longepetiolata,
Euphorbia longeramosa, Euphorbia longinsulicola, Euphorbia
longipila, Euphorbia lupulina, Euphorbia lurida, Euphorbia
lycioides, Euphorbia macropodoides, macvaughiana, Euphorbia manca,
Euphorbia mandoniana, Euphorbia mangleti, Euphorbia mango,
Euphorbia marylandica, Euphorbia mayana, Euphorbia melanadenia,
Euphorbia melanocarpa, Euphorbia meridensis, Euphorbia mertonii,
Euphorbia mexiae, Euphorbia microcephala, Euphorbia microclada,
Euphorbia micromera, Euphorbia misella, Euphorbia missurica,
Euphorbia montana, Euphorbia montereyana, Euphorbia multicaulis,
Euphorbia multiformis, Euphorbia multinodis, Euphorbia multiseta,
Euphorbia muscicola, Euphorbia neomexicana, Euphorbia nephradenia,
Euphorbia niqueroana, Euphorbia oaxacana, Euphorbia occidentalis,
Euphorbia odontodenia, Euphorbia olivacea, Euphorbia olowaluana,
Euphorbia opthalmica, Euphorbia ovata, Euphorbia pachypoda,
Euphorbia pachyrhiza, Euphorbia padifolia, Euphorbia palmeri,
Euphorbia paludicola, Euphorbia parciflora, Euphorbia parishii,
Euphorbia parryi, Euphorbia paxiana, Euphorbia pediculifera,
Euphorbia peplidion, Euphorbia peploides, Euphorbia peplus,
Euphorbia pergamena, Euphorbia perlignea, Euphorbia petaloidea,
Euphorbia petaloidea, Euphorbia petrina, Euphorbia picachensis,
Euphorbia pilosula, Euphorbia pilulifera, Euphorbia pinariona,
Euphorbia pinetorum, Euphorbia pionosperma, Euphorbia platysperma,
Euphorbia plicata, Euphorbia poeppigii, Euphorbia poliosperma,
Euphorbia polycarpa, Etuphorbia polycnemoides, Euphorbia
polyphylla, Euphorbia portoricensis, Euphorbia portulacoides
Euphorbia portulana, Euphorbia preslii, Euphorbia prostrata,
Euiphorbia pteroneura, Euphorbia pycnanthema, Euphorbia ramosa,
Euphorbia rapulum, Euphorbia remyi, Euphorbia retroscabra,
Euphorbia revoluta, Euphorbia rivularis, Euphorbia robusta,
Euphorbia romosa, Euphorbia rubida, Euphorbia rubrosperma,
Euphorbia rupicola, Euphorbia sanmartensis, Euphorbia saxatilis M.
Bieb, Euphorbia schizoloba, Euphorbia sclerocyathium, Euphorbia
scopulorum, Euphorbia senilis, Euphorbia serpyllifolia, Euphorbia
serrula, Euphorbia setiloba Engelm, Euphorbia sonorae, Euphorbia
soobyi, Euphorbia sparsiflora, Euphorbia sphaerosperma, Euphorbia
syphilitica, Euphorbia spruceana, Euphorbia subcoerulea, Euphorbia
stellata, Euphorbia submammilaris, Euphorbia subpeltata, Euphorbia
subpubens, Euphorbia subreniforme, Euphorbia subtrifoliata,
Euphorbia succedanea, Euphorbia tamaulipasana, Euphorbia
telephioicles, Euphorbia tenuissima, Euphorbia tetrapora, Euphorbia
tirucalli, Euphorbia tomentella, Euphorbia tomentosa, Euphorbia
torralbasii, Euphorbia tovariensis, Euphorbia trachysperma,
Euphorbia tricolor, Euphorbia troyana, Euphorbia tuerckheimii,
Euphorbia turczaninowii, Euphorbia umbellulata, Eiphorbia undulata,
Euphorbia vermiformis, Euphorbia versicolor, Euphorbia villifera,
Euphorbia violacea, Euphorbia whitei, Euphorbia xanti Engelm,
Euphorbia xylopoda Greenm., Euphorbia yayalesia Urb., Euphorbia
yungasensis, Euphorbia zeravschanica and Euphorbia zinniiflora.
111. A method according to claim 110 wherein the species of
Euphorbia is Euphorbia peplus.
112. A method according to claim 102 wherein the chemical agent is
a jatrophane or a derivative thereof or a pharmaceutically
acceptable salt of these.
113. A method according to claim 112 wherein said derivative is an
ester derivative.
114. A method according to claim 112 wherein said derivative is an
acetylated derivative.
115. A method according to claim 102 wherein said chemical agent is
a pepluane or a derivative thereof or a pharmaceutically acceptable
salt of these.
116. A method according to claim 115 wherein said derivative is an
ester derivative.
117. A method according to claim 115 wherein said derivative is an
acetylated derivative.
118. A method according to claim 102 wherein said chemical agent is
a paraliane or a derivative thereof or a pharmaceutically
acceptable salt of these.
119. A method according to claim 118 wherein said derivative is an
ester derivative.
120. A method according to claim 118 wherein said derivative is an
acetylated derivative.
121. A method according to claim 102 wherein said compound is an
angeloyl-substituted ingenane or a derivative thereof or a
pharmaceutically acceptable salt of these.
122. A method according to claim 121 wherein said derivative is an
acetylated derivative.
123. A method according to claim 121 wherein said jatrophane is of
conformation 2.
124. A method according to claim 112 or 115 or 118 or 121 wherein
the derivative comprises a substitution as represented in any one
of general formulae (I)-(VI).
125. A method according to claim 102 wherein said compound is
5,8,9,10,14-pentaacetoxy-3-benzoyloxy-15-hydroxypepluane (pepluane)
or a derivative thereof or a pharmaceutically acceptable salt of
these.
126. A method according to claim 125 wherein said derivative is an
ester derivative.
127. A method according to claim 102 wherein said compound is
2,3,5,7,15-pentaacetoxy-9-nicotinoyloxy-14-oxojatropha-6(17),11E-diene
(jatrophane 1) or a derivative thereof or a pharmaceutically
acceptable salt of these.
128. A method according to claim 127 wherein said derivative is an
ester derivative.
129. A method according to claim 102 wherein said compound is
2,5,7,8,9,14-hexaacetoxy-3-benzoyloxy-15-hydroxy-jatropha-6(17),11E-diene
(jatrophane 2) or a derivative thereof or a pharmaceutically
acceptable salt of these.
130. A method according to claim 129 wherein said derivative is an
ester derivative.
131. A method according to claim 102 wherein said compound is
2,5,14-triacetoxy-3-benzoyloxy-8,15-dihydroxy-7-isobutyroyloxy-9-nicotino-
yloxyjatropha-6(17), 11E-diene (jatrophane 3) or a derivative
thereof or a pharmaceutically acceptable salt of these.
132. A method according to claim 131 wherein said derivative is an
ester derivative.
133. A method according to claim 102 wherein said compound is
2,5,9,14-tetraacetoxy-3-benzoyloxy-8,15-dihydroxy-7-isobutyroyloxyjatroph-
a-6(17),11E-diene) (jatrophane 4) or a derivative thereof or a
pharmaceutically acceptable salt of these.
134. A method according to claim 133 wherein said derivative is an
ester derivative.
135. A method according to claim 102 wherein said compound is
2,5,7,14-tetraacetoxy-3-benzoyloxy-8,15-dihydroxy-9-nicotinoyloxyjatropha-
-6(17),11E-diene (jatrophane 5) or a derivative thereof or a
pharmaceutically acceptable salt of these.
136. A method according to claim 135 wherein said derivative is an
ester derivative.
137. A method according to claim 102 wherein said compound is
2,5,7,9,14-pentaacetoxy-3-benzoyloxy-8,15-dihydroxyjatropha-6(17),11E-die-
ne (jatrophane 6) or a derivative thereof or a pharmaceutically
acceptable salt of these.
138. A method according to claim 137 wherein said derivative is an
ester derivative.
139. A method according to claim 102 wherein said compound is
20-O-acetyl-ingenol-3-angelate or a derivative thereof or a
pharmaceutically acceptable salt of these.
140. A method according to claim 139 wherein said derivative is an
ester derivative.
141. A method according to claim 112 or 115 or 118 or 121 or 125 or
127 or 129 or 131 or 133 or 135 or 137 or 139 wherein said compound
is provided in the form of a composition comprising a
pharmaceutically- or cosmetically-acceptable carrier.
142. A method according to claim 141 wherein said carrier is
selected from .beta.-alanine betaine hydrochloride and
t-4-hydroxy-N,N-dimethylproline.
143. A computer program product for assessing the likely usefulness
of a candidate compound or group of compounds for the treatment or
prophylaxis of a PKC-related condition or disorder in a subject,
said product comprising: (1) code that receives as input index
values for at least two features associated with said compound(s),
wherein said features are selected from: (a) the ability to
modulate PKC activity or effect; (b) the ability to induce bipolar
dendritic activity; (c) the ability to be derived from a member of
the Euphorbiaceae family; (d) the ability to be derived from E.
peplus; (e) the ability to be water extractable from the sap of a
Euphorbia species; (f) the ability to activate latent virus; or (g)
less tumor promoting capacity than TPA or MPA; (2) code that adds
said index values to provide a sum corresponding to a potency value
for said compound(s); and (3) a computer readable medium that
stores the codes.
144. A computer for assessing the likely usefulness of a candidate
compound or group of compounds for the treatment of a PKC-related
conditinon or disorder in a subject, wherein said computer
comprises: (1) a machine-readable data storage medium comprising a
data storage material encoded with machine-readable data, wherein
said machine-readable data comprise index values for at least two
features associated with said compound(s), wherein said features
are selected from: (a) the ability to modulate PKC activity or
effect; (b) the ability to induce bipolar dendritic activity; (c)
the ability to be derived from a member of the Euphorbiaceae
family; (d) the ability to be derived from E. peplus; (e) the
ability to be water extractable from the sap of a Euphorbia
species; (h) the ability to activate latent virus; or (i) less
tumor promoting capacity than TPA or MPA; (2) a working memory for
storing instructions for processing said machine-readable data; (3)
a central-processing unit coupled to said working memory and to
said machine-readable data storage medium, for processing said
machine readable data to provide a sum of said index values
corresponding to a potency value for said compound(s); and (4) an
output hardware coupled to said central processing unit, for
receiving said potency value.
Description
FIELD OF THE INVENTION
[0001] The present invention relates generally to chemical agents
useful in the treatment and prophylaxis of protein kinase C (PKC)
related conditions in mammals, including humans and primates,
non-mammalian animals and avian species. More particularly, the
present invention provides a chemical agent of the macrocyclic
diterpene family obtainable from a member of the Euphorbiaceac
family of plants or botanical or horticultural relatives thereof or
derivatives or chemical analogues or chemically synthetic forms of
the agents for use in the treatment or prophylaxis of PKC-related
conditions in mammalian, animal and avian subjects. The subject
chemical agents are also useful for modulating expression of
genetic sequences including promotion and other regulatory
sequences. The present invention further contemplates a method for
the prophylaxis and/or treatment in mammalian, animal or avian
subjects with PKC-related conditions by the topical or systemic
administration of a macrocyclic diterpene obtainable from a member
of the Euphorbiaceae family of plants or their botanical or
horticultural derivatives or a derivative, chemical analogue or
chemically synthetic form of the agent. The chemical agent of the
present invention may be in the form of a purified compound,
mixture of compounds, a precursor form of one or more of the
compounds capable of chemical transformation into a therapeutically
and/or genetically active agent or in the form of a chemical
fraction, sub-fraction, preparation or extract of the plant.
BACKGROUND OF THE INVENTION
[0002] Bibliographic details of the publications referred to by
author in this specification are collected at the end of the
description.
[0003] Reference to any prior art in this specification is not, and
should not be taken as, an acknowledgment or any form of suggestion
that this prior art forms part of the common general knowledge in
Australia or any other country.
[0004] Natural product screening is a term applied to the screening
of natural environments for bioactive molecules. Particularly
sought after bioactive molecules are those having potential as
useful therapeutic agents. Natural environments include plants,
microorganisms, coral and marine animals. The search for potential
therapeutic agents for the treatment of cancer and infection by
pathogenic organisms remains an important focus.
[0005] The Euphorbiaceae family of plants covers a wide variety of
plants including weeds of Euphorbia species. There have been a
variety of inconclusive reports on the potential effects of the sap
of these plants on a variety of conditions as well as promoting
tumorigenesis and causing skin and ocular irritation.
[0006] The most intensively studied species of this group is
Euphorbia pilulifera L (synonyms E. hirta L., E. capitata Lam.),
whose common names include pill-bearing spurge, snakeweed, cat's
hair, Queensland asthma weed and flowery-headed spurge. The plant
is widely distributed in tropical countries, including India, and
in Northern Australia, including Queensland.
[0007] A recent report describes selective cytotoxicity of a number
of tiglilane diterpene esters from the latex of Euphorbia poisonii,
a highly toxic plant found in Northern Nigeria, which is used as a
garden pesticide. One of these compounds has a selective
cytotoxicity for the human kidney carcinoma cell line A-498 more
than 10,000 times greater than that of adriamycin (Fatope et al.,
1996).
[0008] Euphorbia hirta plants and extracts thereof have been
considered for a variety of purposes, including tumor therapy (EP 0
330 094), AIDS-related complex and AIDS (HU-208790) and increasing
immunity and as an anti-fungoid agent for treatment of open wounds
(DE-4102054).
[0009] Thus, while there are isolated reports of anti-cancer
activity of various Euphorbia preparations (see Fatope et al.,
1996; Oksuz et al., 1996), not only are the compounds present in at
least one Euphorbia species reported to be carcinogenic (Evans and
Osman, 1974; Stavric and Stolz, 1976; Hecker, 1970), but at least
one species has a skin-irritant and tumor-promoting effect (Gundidz
et al., 1993) and another species reduces EBV-specific cellular
immunity in Burkitt's lymphoma (Imai, 1994).
[0010] In accordance with the present invention, the inventors have
identified chemical agents and fractions comprising these agents
which are useful in the treatment and prophylaxis of PKC-related
conditions in an animal, mammal or avian species. The instant
agents are also useful in modulating and in particular stimulating
or otherwise promoting expression or function of nucleotide
sequences such as promoter or other regulatory sequences which is
useful in gene therapy, genetic therapy, genetic augmentation and
gene replacement therapies as well as promoting expression of
genetic material into an expression product.
SUMMARY OF THE INVENTION
[0011] Throughout this specification, unless the context requires
otherwise, the word "comprise", or variations such as "comprises"
or "comprising", will be understood to imply the inclusion of a
stated element or integer or group of elements or integers but not
the exclusion of any other element or integer or group of elements
or integers.
[0012] The present invention is predicated in part on the
identification of chemical agents and fractions comprising same
from plants of the Euphorbiaceae family which are useful in the
treatment and prophylaxis of PKC-related conditions in an animal,
mammal incluidng a human and avian species. The inventors have
identified that the chemical agents of the present invention are
capable of modulating protein kinase C (PKC) activity thus
providing a basis for the treatment of conditions where PKC
activity is required to be up-regulated or down-regulated. The
instant chemical agents further modulate and more particularly
activate or otherwise promote expression of genetic sequences
and/or operation of promoter or other regulatory sequences. The
latter is useful for genetic therapies including gene therapy.
[0013] Accordingly, one aspect of the present invention
contemplates a method for the treatment or prophylaxis of a
PKC-related condition or disorder in a subject, said method
comprising the administration to said subject of a
symptom-ameliorating effective amount of a chemical agent
obtainable from a plant of the Euphorbiaceae family or a derivative
or chemical analogue thereof which chemical agent is a macrocyclic
diterpene selected from compounds of the ingenane, pepluane and
jatrophane families and which chemical agent or derivative or
chemical analogue is represented by any one of the general formulae
(I)-(V) as defined herein and which chemical agent or derivative or
chemical analogue thereof is capable of modulating PKC activity,
PKC-gene expression or PKC enzyme turnover and wherein said
chemical agent or its derivatives or chemical analogues is
administered for a time and under conditions sufficient to
ameliorate one or more symptoms associated with a PKC-related
condition or disorder.
[0014] Another aspect of the present invention provide a method for
the treatment or prophylaxis of a PKC-related condition or disorder
in a subject, said method comprising the administration to said
subject of a symptom-ameliorating effective amount of a macrocyclic
diterpene, or a chemical fraction comprising same from a plant of
the family Euphorbiaceae or a derivative or chemical analogue of
said macrocyclic diterpene having the structures as defined above
wherein said macrocyclic diterpene or its derivative or chemical
analogue modulates PKC activity, synthesis or enzyme turnover, said
administration being for a time and under conditions sufficient to
ameliorate one or more symptoms of associated with a PKC-related
condition or disorder.
[0015] Yet another aspect of the invention encompasses a method of
modulating the expression of a genetic sequence for the treatment
or prophylaxis of a condition or disorder in a subject, said
genetic sequence being under the control of a promoter whose
activity is modulated by a chemical agent obtainable from a plant
of the Euphorbiaceae family or a derivative or chemical analogue
thereof which chemical agent is a macrocyclic diterpene selected
from compounds of the ingenane, pepluane and jatrophane families
and which chemical agent or derivative or chemical analogue is
represented by any one of the general formulae (I)-(V) as defined
herein and which chemical agent or derivative or chemical analogue
thereof is capable of modulating PKC activity, PKC-gene expression
or PKC enzyme turnover, said method comprising administering to
said subject an expression facilitating amount of said chemical
agent or derivative or chemical analogue thereof for a time and
under conditions sufficient to facilitate the expression of said
genetic sequence to thereby ameliorate one or more symptoms
associated with said condition or disorder.
[0016] Still another aspect in the invention envisions a method of
modulating the expression of a genetic sequence by the
administration of a chemical agent obtainable from a plant of the
Euphorbiaceae family or a derivative or chemical analogue thereof
which chemical agent is a macrocyclic diterpene selected from
compounds of the ingenane, pepluane and jatrophane familes and
which chemical agent or derivative or chemical analogue is
represented by any one of the general formulae (I)-(V) as defined
herein and which chemical agent or derivative or chemical analogue
thereof is capable of modulating PKC activity, PKC-gene expression
or PKC enzyme turnover and wherein said chemical agent or its
derivatives or chemical analogues is administered for a time and
under conditions sufficient to facilitate the expression of said
genetic sequence.
[0017] Still yet another aspect of the present invention features a
method of stimulating the activation or function of a promoter by
the administration of a chemical agent obtainable from a plant of
the Euphorbiaceae family or a derivative or chemical analogue
thereof which chemical agent is a macrocyclic diterpene selected
from compounds of the ingenane, pepluane and jatrophane families
and which chemical agent or derivative or chemical analogue is
represented by any one of the general formulae (I)-(V) as defined
herein and which chemical agent or derivative or chemical analogue
thereof is capable of modulating PKC activity, PKC-gene expression
or PKC enzyme turnover and wherein said chemical agent or its
derivatives or chemical analogues is administered for a time and
under conditions sufficient to stimulate the activation or function
of said promoter.
[0018] Even yet another aspect of the present invention
contemplates a method for the treatment or prophylaxis of a
PKC-related condition in a subject, said method comprising the
administration to said subject of a symptom-ameliorating or
immunopotentiating effective amount of a macrocyclic diterpene
obtainable from a Euphorbiaceae plant or its botanical or
horticultural relative, said macrocyclic diterpene being selected
from an ingenane, pepluane or jatrophane, or a derivative or
chemical analogue thereof, having the structure represented by any
one of the general formulae (I)-(V) as defined herein and which
chemical agent or derivative or chemical analogue thereof is
capable of modulating PKC activity, PKC-gene expression or PKC
enzyme turnover and wherein said chemical agent or its derivatives
or chemical analogues is administered for a time and under
conditions sufficient to ameliorate one or more symptoms associated
with said biological entity and wherein said chemical agent
exhibits a potency of agent (P.sub.A) of >10, wherein the
P.sub.A=.SIGMA.I.sub.V where I.sub.V is a numerical value
associated with a particular feature as defined in Table A or
pharmaceutically acceptable salts of these, said chemical agent
being administered for a time and under conditions sufficient to
ameliorate at least one symptom caused by or associated with said
PKC-related condition.
BRIEF DESCRIPTION OF THE FIGURES
[0019] FIG. 1 shows the activation of PKC, using a fluorescent
peptide assay ("PepTag" non-radioactive protein kinase kit,
Promega). Lane 1, PKC and substrate alone; lane 2, plus positive
control activator; lane 3, plus 100 ng/ml TPA; lane 4, plus 0.1
ng/ml TPA; lane 5, plus 0.01 ng/ml TPA; lane 6, plus 0.001 ng/ml
TPA; lane 7, ether extract of E. peplus sap in DEM, diluted 1 in 5;
lane 8, aqueous layer from ether extraction, diluted 1/25; lane 9,
crude sap diluted 1/25; lane 10, DME alone.
[0020] FIG. 2 shows the activation of PKC by E. peplus fractions.
Lanes 1 and 2, same as FIG. 1; lane 3, 2 mg/ml fraction H; lane 4,
2 mg/ml ingenanes.
[0021] FIG. 3 is photographic representation showing the results of
a PKC assay using rat brain PKC. Lane 1, negative control; lane 2,
positive control; lane 3, empty; lane 4, PEP001 (1/125 dilution),
lane 5, PEP001 (1/500 dilution) and lane 6, TPA (20 .mu.g).
[0022] FIG. 4 is a photographic representation showing the
activation of PKC in MM96L cells expressing PKC fused to green
fluorescent protein (GFP). (A) PKC.beta. expressed in the nuclei of
MM96L human melanoma PKC MM96L cells in the absence of drug. (B)
After treatment with crude E. peplus extract for 2 hr.
[0023] FIG. 5 is a photographic representation showing induction of
translocation of activated PKCs by the compounds of the instant
invention to the cytoplasm, plasma membrane and to the Golgi or
similarly located cellular structure.
[0024] FIG. 6 is a graphical representation showing the induction
of translation of the classical and novel PKC isoforms in response
to PEP003, PEP005, bryostatin-1 and TPA.
[0025] FIG. 7 is a graphical representation showing the activation
of HIV from U1 cells.
[0026] FIG. 8 is a graphical representation showing treatment of
lytic HIV infection of peripheral blood mononuclear cells (PBMC)
with PEP003, PEP004, TPA and ingenol, expressed as p24 production
over a 10 day treatment period. (A) Uninfected cells, (B) low titer
infected cells, (C) low titer infected cells represented as p24
production versus drug concentration, (D) same as (C) but high
titer infection.
[0027] FIG. 9 is a photographic representation showing the
recruitment of neutrophils in the skin induced by PEP001 extract.
(A) Normal skin of nude mouse. (B) Skin of nude mouse showing
infiltration of neutrophils one day after treatment with E. peplus
sap.
[0028] FIG. 10 is a photographic representation showing effect of
PEP010 onrecruitment of neutrophils in normal skin of nude mouse
and skin overlying subcutaneously implanted B16 melanoma. (A) 24 hr
treatment, (B) 48 hr treatment.
[0029] FIG. 11 is a graphical representation illustrating the
ability of PEP001 to induce the release of superoxide radical, as
demonstrated by fluorescence-activated cell sorting.
[0030] FIG. 12 is a graphical representation showing the effect of
pre-treatment of leukocytes with PEP003 on E. coli activity (16 hr
incubation), relative to PBS control; depicted as numbers of E.
coli cells/ml media.
[0031] FIG. 13 is a graphical representation showing the effect of
pre-treatment of leukocytes with PEP003 on E. coli numbers depicted
in terms of turbidity.
[0032] FIG. 14 is a photographic representation showing production
of viral capsid antigen (VCA) in B95-8 (EBV+ Marmoset cell line)
after treatment with TPA, PEP003 and PEP004 for 3 and 7 days.
[0033] FIG. 15 is a photographic representation showing production
of viral capsid antigen (VCA) in BL74 and Mutu I (Burkitts lymphoma
cell lines) after treatment with TPA, PEP003 and PEP004 for 3 and 7
days.
[0034] FIG. 16 is a photographic representation showing production
of BZLF1 (the initial transactivator of EBV) after treatment with
TPA, PEP003 and PEP004 for 3 and 7 days.
[0035] FIG. 17 is a graphical representation showing activation of
natural killer cell activity, assayed as % specific lysis of K562
cells (a natural killer-sensitive cell line) after pre-treatment of
AO2-M melanoma cells with PEP003 and TPA.
[0036] FIG. 18 is a graphical representation showing survival of
Jam cells after treatment with saps from the Euphorbiaceae,
expressed as percentage cell survival determined by sulfurhodamine
B staining of cells.
[0037] FIG. 19 is a diagrammatic representation of a system used to
carry out the instructions encoded by the storage medium of FIGS. 9
and 10.
[0038] FIG. 20 is a diagrammatic representation of a cross-section
of a magnetic storage medium.
[0039] FIG. 21 is a diagrammatic representation of a cross-section
of an optically readable data storage system.
[0040] Compounds may be referred to in the subject specification by
a compound code. These are defined as below:
1 TABLE OF COMPOUND CODES COMPOUND CODE DESCRIPTION PEP001 Crude
sap PEP002 Methanol and ether extract of E. peplus sap prepared
according to Example 7 of PCT/AU98/00656 PEP003 Ingenane enriched
fraction prepared according to Examples 21 and 23 PEP004
Jatrophane/Pepluane enriched fraction prepared according to Example
7 of PCT/AU98/00656 PEP005 20-hydroxy-ingenol-3-angelate PEP006
Ingenol-3-angelate PEP008 20-O-acetyl-ingenol-3-angelate PEP009
Acetone Extract of XAD prepared according to Example 21 PEP010
Ingenane enriched fraction prepared according to Examples 22 and
23
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0041] The present invention is predicated in part on the
identification of biologically useful properties of chemical agents
and chemical fractions comprising these agents obtainable from a
member of the Euphorbiaceae family of plants or their botanical or
horticultural relatives. These biologically useful properties
include their use in the prophylaxis and/or treatment of
PKC-related conditions. Reference herein to a "PKC-related
condition" includes a PKC-related disorder and comprises medical
including psychological conditions associated with aberrant PKC
activity including activating low PKC activity or lowering higher
than normal levels of PKC as well as conditions associated with the
presence of latent virus in a host cell.
[0042] Examples of PKC disorders include but are not limnited to
alcoholism, Alzheimer's disease, asthma, atherosclosis, atopic
dermatitis, autoimmune disease, bipolar-disorder, blood disorder,
cardiac hypertrophy, depression, diabetes, hypertension,
hyperplastic dermatosis, multiple sclerosis, myocardial ischemia,
osteoarthritis, psoriasis, rheumatoid arthritis and
transplantation. The instant chemical agents are also useful in
promoting gene activation and promotion.
[0043] The term "treatment" is used in its broadest sense and
includes the prevention of a PKC-related condition or the reversal
of a condition to asymptomatic levels or to levels in between as
well as facilitating the amelioration of the effects of symptoms of
the PKC-related condition. A PKC-related condition occurs when an
increase or decrease in PKC promotes or facilitates an effect.
Furthermore, "treatment" further encompasses promoting an elevation
in promoter or other regulatory sequence activity and/or structural
gene sequence expression.
[0044] The term "prophylaxis" is also used herein in its broadest
sense to encompass a reduction in the risk of development of a
PKC-related condition. In certain conditions, an agent may act to
treat a subject prophylactically. Furthermore, the prophylactic
administration of an agent may result in the agent becoming
involved in the treatment of a pathological condition. Use of the
terms "treatment" or "prophylaxis" is not to be taken as limiting
the intended result which is to reduce the incidence of a
PKC-related disorder or condition and/or to ameliorate the symptoms
or risk of development of symptoms caused or facilitated by a
PKC-related condition.
[0045] The present invention is particularly directed to the use of
one or more macrocyclic diterpenes from a member of the
Euphorbiaceae family of plants or botanical or horticultural
relatives of such plants. Reference herein to a member of the
Euphorbiaceae family includes reference to species from the genera
Acalypha, Acidoton, Actinostemon, Adelia, Adenocline, Adenocrepis,
Adenophaedra, Adisca, Agrostistachys, Alchornea, Alchorneopsis,
Alcinaeanthus, Alcoceria, Aleurites, Amanoa, Andrachne, Angostyles,
Anisophyllum, Antidesma, Aphora, Aporosa, Aporosella, Argythamnia,
Astrococcus, Astrogyne, Baccanrea, Baliospermum, Bernardia,
Beyeriopsis, Bischofia, Blachia, Blumeodondron, Bonania, Bradleia,
Breynia, Breyniopsis, Briedelia, Buraeavia, Caperonia,
Caryodendron, Celianella, Cephalocroton, Chaenotheca, Chaetocarpus,
Chamaesyce, Cheilosa, Chiropetalum, Choriophyllum, Cicca,
Chaoxylon, Cleidon, Cleistanthus, Cluytia, Cnesmone, Cnidoscolus,
Coccoceras, Codiaeum, Coelodiscus, Conami, Conceveiba,
Conceveibastrum, Conceveibum, Corythea, Croizatia, Croton,
Crotonopsis, Crozophora, Cubanthus, Cunuria, Dactylostemon,
Dalechampia, Dendrocousinsia, Diaspersus, Didymocistus,
Dimorphocalyx, Discocarpus, Ditaxis, Dodecastingma, Drypetes,
Dysopsis, Elateriospermum, Endadenium, Endospermum, Erismanthus,
Erythrocarpus, Erythrochilus, Eumecanthus, Euphorbia,
Euphorbiodendron, Excoecaria, Flueggea, Calearia, Garcia,
Gavarretia, Gelonium, Giara, Givotia, Glochidion, Clochidionopsis,
Glycydendron, Gymnanthes, Gymnosparia, Haematospermum, Hendecandra,
Hevea, Hieronima, Hieronyma, Hippocrepandra, Homalanthus,
Hymenocardia, Janipha, Jatropha, Julocroton, Lasiocroton,
Leiocarpus, Leonardia, Lepidanthus, Leucocroton, Mabea, Macaranga,
Mallotus, Manihot, Mappa, Maprounea, Melanthesa, Mercurialis,
Mettenia, Micrandra, Microdesmis, Microelus, Microstachy,
Maocroton, Monadenium, Mozinna, Neoscortechinia, Omalanthus,
Omphalea, Ophellantha, Orbicularia, Ostodes, Oxydectes, Palenga,
Pantadenia, Paradrypeptes, Pausandra, Pedilanthus, Pera, Peridium,
Petalostigma, Phyllanthus, Picrodendro, Pierardia, Pilinophytum,
Pimeleodendron, Piranhea, Platygyna, Plukenetia, Podocalyx,
Poinsettia, Poraresia, Prosartema, Pseudanthus, Pycnocoma,
Quadrasia, Reverchonia, Richeria, Richeriella, Ricinella,
Ricinocarpus, Rottlera, Sagotia, Sanwithia, Sapium, Savia,
Sclerocroton, Sebastiana, Securinega, Senefeldera, Senefilderopsis,
Serophyton, Siphonia, Spathiostemon, Spixia, Stillingia,
Strophioblachia, Synadenium, Tetracoccus, Tetraplandra,
Tetrorchidium, Thyrsanthera, Tithymalus, Trageia, Trewia,
Trigonostemon, Tyria and Xylophylla.
[0046] The most preferred genus and most suitable for the practice
of the present invention is the genus Euphorbia. Particularly
useful species of this genus include Euphorbia aaron-rossii,
Euphorbia abbreviata, Euphorbia acuta, Euphorbia alatocaulis,
Euphorbia albicaulis, Euphorbia algomarginata, Euphorbia aliceae,
Euphorbia alta, Euphorbia anacampseros, Euphorbia andromedae,
Euphorbia angusta, Euphorbia anthonyi, Euphorbia antiguensis,
Euphorbia apocynifolia, Euphorbia arabica, Euphorbia ariensis,
Euphorbia arizonica, Euphorbia arkansana, Euphorbia arteagae,
Euphorbia arundelana, Euphorbia astroites, Euphorbia atrococca,
Euphorbia baselicis, Euphorbia batabanensis, Euphorbia bergeri,
Euphorbia bermudiana, Euphorbia bicolor, Euphorbia biformis,
Euphorbia bifurcata, Euphorbia bilobata, Euphorbia biramensis,
Euphorbia biuncialis, Euphorbia blepharostipula, Euphorbia
blodgetti, Euphorbia boerhaavioides, Euphorbia boliviana, Euphorbia
bracei, Euphorbia brachiata, Euphorbia brachycera, Euphorbia
brandegee, Euphorbia brittonii, Euphorbia caesia, Euphorbia
calcicola, Euphorbia campestris, Euphorbia candelabrum, Euphorbia
capitellata, Euphorbia carmenensis, Euphorbia carunculata,
Euphorbia cayensis, Euphorbia celastroides, Euphorbia chalicophila,
Euphorbia chamaerrhodos, Euphorbia chamaesula, Euphorbia
chiapensis, Euphorbia chiogenoides, Euphorbia cinerascens,
Euphorbia clarionensis, Euphorbia colimae, Euphorbia colorata,
Euphorbia commutata, Euphorbia consoquitlae, Euphorbia
convolvuloides, Euphorbia corallifera, Euphorbia creberrima,
Euiphorbia crenulata, Euphorbia cubensis, Euphorbia cuspidata,
Euphorbia cymbiformis, Euphorbia darlingtonii, Euphorbia defoliata,
Euphorbia degeneri, Euphorbia deltoidea, Euphorbia dentata,
Euphorbia depressa Euphorbia dictyosperma, Euphorbia dictyosperma,
Euphorbia dioeca, Euphorbia discoidalis, Euphorbia dorsiventralis,
Euphorbia drumondii, Euphorbia duclouxii, Euphorbia dussii,
Euphorbia eanophylla, Euphorbia eggersii, Euphorbia eglandulosa,
Euphorbia elata, Euphorbia enalla, Eiphorbia eriogonoides,
Euphorbia eriophylla, Euphorbia esculaeformis, Euphorbia
espirituensis, Euphorbia esula, Euphorbia excisa, Euphorbia
exclusa, Euphorbia exstipitata, Euphorbia exstipulata, Euphorbia
fendleri, Euphorbia filicaulis, Euphorbia filiformis, Euphorbia
florida, Euphorbia fruticulosa, Euphorbia garber, Euphorbia
gaumerii, Euphorbia gerardiana, Euphorbia geyeri, Euphorbia
glyptosperma, Euphorbia gorgonis, Euphorbia gracilior, Euphorbia
gracillima, Euphorbia gradyi, Euphorbia graminea, Euphorbia
graminiea Euphorbia grisea, Euphorbia guadalajarana, Euphorbia
guanarensis, Euphorbia gymnadenia, Euphorbia haematantha, Euphorbia
hedyotoides, Euphorbia heldrichii, Euphorbia helenae, Euphorbia
helleri, Euphorbia helwigii, Euphorbia henricksonii, Euphorbia
heterophylla, Euphorbia hexagona, Euphorbia hexagonoides, Euphorbia
hinkleyorum, Euphorbia hintonii, Euphorbia hirtula, Euphorbia
hirta, Euphorbia hooveri, Euphorbia humistrata, Euphorbia
hypericifolia, Euphorbia inundata, Euphorbia involuta, Euphorbia
jaliscensis, Euphorbia jejuna, Euphorbia johnston, Euphorbia
juttae, Euphorbia knuthii, Euphorbia lasiocarpa, Euphorbia lata,
Euphorbia latazi, Euphorbia latericolor, Euphorbia laxiflora
Euphorbia lecheoides, Euphorbia ledienii, Euphorbia leucophylla,
Euphorbia lineata, Euphorbia linguiformis, Euphorbia longecornuta,
Euphorbia longepetiolata, Euphorbia longeramosa, Euphorbia
longinsulicola, Euphorbia longipila, Euphorbia lupulina, Euphorbia
lurida, Euphorbia lycioides, Euphorbia macropodoides, macvaughiana,
Euphorbia manca, Euphorbia mandoniana, Euphorbia mangleti,
Euphorbia mango, Euphorbia marylandica, Euphorbia mayana, Euphorbia
melanadenia, Euphorbia melanocarpa, Euphorbia meridensis, Euphorbia
mertonii, Euphorbia mexiae, Euphorbia microcephala, Euphorbia
microclada, Euphorbia micromera, Euphorbia misella, Euphorbia
missurica, Euphorbia montana, Euphorbia montereyana, Euphorbia
multicaulis, Euphorbia multiformis, Euphorbia multinodis, Euphorbia
multiseta, Euphorbia muscicola, Euphorbia neomexicana, Euphorbia
nephradenia, Euphorbia niqueroana, Euphorbia oaxacana, Euphorbia
occidentalis, Euphorbia odontodenia, Euphorbia olivacea, Euphorbia
olowaluana, Euphorbia opthalmica, Euphorbia ovata, Euphorbia
pachypoda, Euphorbia pachyrhiza, Euphorbia padifolia, Euphorbia
palmeri, Euphorbia paludicola, Euphorbia parciflora, Euphorbia
parishii, Euphorbia parryi, Euphorbia paxiana, Euphorbia
pediculifera, Euphorbia peplidion, Euphorbia peploides, Euphorbia
peplus, Euphorbia pergamena, Euphorbia perlignea, Euphorbia
petaloidea, Euphorbia petaloidea, Euphorbia petrina, Euphorbia
picachensis, Euphorbia pilosula, Eiphorbia pilulifera, Euphorbia
pinariona, Euphorbia pinetorum, Euphorbia pionosperma, Euphorbia
platysperma, Euphorbia plicata, Euphorbia poeppigii, Euphorbia
poliosperma, Euphorbia polycarpa, Euphorbia polycnemoides,
Euphorbia polyphylla, Euphorbia portoricensis, Euphorbia
portulacoides Euphorbia portulana, Euphorbia preslii, Euphorbia
prostrata, Euphorbia pteroneura, Euphorbia pycnanthema, Euphorbia
ramosa, Euphorbia rapulum, Euphorbia remyi, Euphorbia r etroscabra,
Euphorbia revoluta, Euphorbia rivularis, Euphorbia robusta,
Euphorbia romosa, Euphorbia rubida, Euphorbia rubrosperma,
Euphorbia rupicola, Euphor bia sanmartensis, Euphorbia saxatilis M.
Bieb, Euphorbia schizoloba, Euphorbia sclerocyathium, Euphorbia
scopulorum, Euphorbia senilis, Euphorbia serpyllifolia, Euphorbia
serrula, Euphorbia setiloba Engelm, Euphorbia sonorae, Euphorbia
soobyi, Euphorbia sparsiflora, Euphorbia sphaerosperma, Euphorbia
syphilitica, Euphorbia spruceana, Euphorbia subcoerulea, Euphorbia
stellata, Euphorbia submammilaris, Euphorbia subpeltata, Euphorbia
subpubens, Euphorbia subreniforme, Euphorbia subtrifoliata,
Euphorbia succedanea, Euphorbia tamaulipasana, Euphorbia
telephioides, Euphorbia tenuissima, Euphorbia tetrapora, Euphorbia
tirucalli, Euphorbia tomentella, Euphorbia tomentosa, Euphorbia
torralbasii, Euphorbia tovariensis, Euphorbia trachysperma,
Euphorbia tricolor, Euphorbia troyana, Euphorbia tuerckheimii,
Euphorbia turczaninowii, Euphorbia umbellulata, Euphorbia undulata,
Euphorbia vermiformis, Euphorbia versicolor, Euphorbia villifera,
Euphorbia violacea, Euphorbia whitei, Euphorbia xanti Engelm,
Euphorbia xylopoda Greenm., Euphorbia yayalesia Urb., Euphorbia
yungasensis, Euphorbia zeravschanica and Euphorbia zinniiflora.
[0047] Particularly preferred species of the genus Synadenium
include Synadenium grantii and Synadenium compactum.
[0048] Particularly preferred species of the genus Monadenium
include Monadenium lugardae and Monadenium guentheri.
[0049] A preferred species of the genus Endadenium is Endadenium
gossweileni.
[0050] Euphorbia peplus is particularly useful in the practice of
the present invention. Reference herein to "Euphorbia peplus" or
its abbreviation "E. peplus" includes various varieties, strains,
lines, hybrids or derivatives of this plant as well as its
botanical or horticultural relatives. Furthermnore, the present
invention may be practiced using a whole Euphorbiaceae plant or
parts thereof including sap or seeds or other reproductive material
may be used. Generally, for seeds or reproductive material to be
used, a plant or plantlet is first required to be propagated.
[0051] Reference herein to a Euphorbiaceae plant, a Euphorbia
species or E. peplus further encompasses genetically modified
plants. Genetically modified plants include trangenic plants or
plants in which a trait has been removed or where an endogenous
gene sequence has been down-regulated, mutated or otherwise altered
including the alteration or introduction of genetic material which
exhibits a regulatory effect on a particular gene. Consequently, a
plant which exhibits a character not naturally present in a
Euphorbiaceae plant or a species of Euphorbia or in E. peplus is
nevertheless encompassed by the present invention and is included
within the scope of the above-mentioned terms.
[0052] The macrocyclic diterpenes are generally in extracts of the
Euphorbiaceae plants. An extract may comprise, therefore, sap or
liquid or semi-liquid material exuded from, or present in, leaves,
stem, flowers, seeds, bark or between the bark and the stem. Most
preferably, the extract is from sap. Furthermore, the extract may
comprise liquid or semi-liquid material located in fractions
extracted from sap, leaves, stems, flowers, bark or other plant
material of the Euphoriaceae plant. For example, plant material may
be subject to physical manipulation to disrupt plant fibres and
extracellular matrix material and inter- and intra-tissue extracted
into a solvent including an aqueous environment. All such sources
of the macrocyclic diterpenes are encompassed by the present
invention including macrocyclic diterpenes obtained by synthetic
routes.
[0053] The preferred macrocyclic diterpenes are selected from
compounds of the ingenane, pepluane and jatrophane families. A
compound is stated to be a member of the ingenane, pepulane or
jatrophane families on the basis of chemical structure and/or
chemical or physical properties. A compound which is a derivative
of an ingenane, pepluane or jatrophane is nevertheless encompassed
by the present invention through use of the terms "ingenane",
"pepluane" or "jatrophane" since these terms include derivatives,
chemical analogues and chemically synthetic forms of these families
of compounds. One particularly preferred derivative is an angeloyl
derivative of ingenane.
[0054] The preferred chemical agent of the present invention is one
which exhibits an effect on PKC. Such an effect may be a direct
activation or inhibition of PKC activity or a direct effect on the
levels of PKC enzyme in a cell or exported from a cell.
Furthermore, the effect may be transitory or may involve an initial
activation of PKC activity or PKC enzyme synthesis or induction of
a functional conformation followed by a down-regulation of PKC
activity, enzyme levels or formation of a deactivated conformation.
Consequently, an effect on PKC is regarded herein as a modulatory
effect and is conveniently determined by consequential events such
as resulting from altered signal transduction. For example,
activation of latent virus, activation of immune mechanisms or
activation of a gene promoter may occur and this is regarded herein
as a modulatory effect on PKC.
[0055] The chemical agents of the present invention may be in
purified or isolated form meaning that the preparation is
substantially devoid of other compounds or contaminating agents
other than diluent, solvent or carrier or isoforms of the agents.
Furthermore, the term "chemical agent" includes preparations of two
or more compounds either admixed together or co-purified from a
particular source. The chemical agent may also be a chemical
fraction, extract or other preparation from the Euphorbiaceace
plant.
[0056] Consequently, reference herein to a "chemical agent"
includes a purified form of one or more compounds or a chemical
fraction or extract such as from the sap of a Euphorbiaceace plant,
and in particular a species of Euphorbia, and most preferably from
E. peplus or botanical or horticultural relatives or variants
thereof.
[0057] Accordingly, one aspect of the present invention
contemplates a method for the treatment or prophylaxis of a
PKC-related condition or disorder in a subject, said method
comprising the administration to said subject of a
symptom-ameliorating effective amount of a chemical agent
obtainable from a plant of the Euphorbiaceae family or a derivative
or chemical analogue thereof which chemical agent is a macrocyclic
diterpene selected from compounds of the ingenane, pepluane and
jatrophane families and which chemical agent or derivative or
chemical analogue is represented by any one of the general formulae
(I)-(V) 1
[0058] wherein:
[0059] n is 0-10 atoms selected from carbon, oxygen, nitrogen,
sulfur, phosphorus, silicon, boron, arsenic and selenium, wherein
the ring defined by said atoms is saturated or unsaturated,
including epoxides and thioepoxides;
[0060] A-T are independently selected from hydrogen, R.sub.1,
R.sub.2, R.sub.3, F, Cl, Br, I, CN, OR.sub.1, SR.sub.1,
NR.sub.1R.sub.2, N(.dbd.O).sub.2, NR.sub.1OR.sub.2,
ONR.sub.1R.sub.2, SOR.sub.1, SO.sub.2R.sub.1, SO.sub.3R.sub.1,
SONR.sub.1R.sub.2, SO.sub.2NR.sub.1R.sub.2,
SO.sub.3NR.sub.1R.sub.2, P(R.sub.1).sub.3,
P(.dbd.O)(R.sub.1).sub.3, Si(R.sub.1).sub.3, B(R.sub.1).sub.2,
(C.dbd.X)R.sub.3 or X(C.dbd.X)R.sub.3 where X is selected from
sulfur, oxygen and nitrogen;
[0061] R.sub.1 and R.sub.2 are each independently selected from
C.sub.1-C.sub.20 alkyl (branched and/or straight chained),
C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8 cycloalkyl,
C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and/or straight chained), C.sub.2-C.sub.10 alkynyl (branched and/or
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.1, SR.sub.1, NR.sub.1R.sub.2, N(.dbd.O).sub.2,
NR.sub.1OR.sub.2, ONR.sub.1R.sub.2, SOR.sub.1, SO.sub.2R.sub.1,
SO.sub.3R.sub.1, SONR.sub.1R.sub.2, SO.sub.2NR.sub.1R.sub.2,
SO.sub.3NR.sub.1R.sub.2, P(R.sub.1).sub.3,
P(.dbd.O)(R.sub.1).sub.3, Si(R.sub.1).sub.3,
B(R.sub.1).sub.2]alkyl;
[0062] R.sub.3 is selected from R.sub.1, R.sub.2, CN, COR.sub.1,
CO.sub.2R.sub.1, OR.sub.1, SR.sub.1, NR.sub.1R.sub.2,
N(.dbd.O).sub.2, NR.sub.1OR.sub.2, ONR.sub.1R.sub.2, SOR.sub.1,
SO.sub.2R.sub.1, SO.sub.3R.sub.1, SONR.sub.1R.sub.2,
SO.sub.2NR.sub.1R.sub.2, SO.sub.3NR.sub.1R.sub.2, P(R.sub.1).sub.3,
P(.dbd.O)(R.sub.1).sub.3, Si(R.sub.1).sub.3, B(R.sub.1).sub.2;
[0063] A connected to B (or C), D (or E, R (or Q), P (or O) or S
(or T) is a selection of C.sub.1-C.sub.8 disubstituted (fused)
saturated or unsaturated carbocyclic or heterocyclic rings further
substituted by R.sub.3, (C.dbd.X)R.sub.3 and X(C.dbd.X)R.sub.3,
including epoxides and thioepoxides;
[0064] J connected to I (or H), G (or F), K (or L), M (or N) or S
(or T) is a selection of C.sub.1-C.sub.8 disubstituted (fused)
saturated and unsaturated carbocyclic or heterocyclic rings further
substituted by R.sub.3, (C.dbd.X)R.sub.3 and X(C.dbd.X)R.sub.3,
including epoxides and thioepoxides;
[0065] D (or E) connected to B (or C) or G (or F); I (or H)
connected to G (or F); P (or O) connected to R (or Q) or M (or N);
K (or L) connected to N (or M) is a selection of C.sub.1-C.sub.8
disubstituted (fused) saturated or unsaturated carbocyclic or
heterocyclic rings substituted by R.sub.3, (C.dbd.X)R.sub.3 and
X(C.dbd.X)R.sub.3, including epoxides and thioepoxides;
[0066] B and C, D and E, R and Q, P and O, I and H, G and F, K and
L, M and N or S and T are .dbd.X where X is selected from sulfur,
oxygen, nitrogen, NR.sub.1R.sub.2, and .dbd.CR.sub.1R.sub.2 2
[0067] wherein:
[0068] n is 0-10 atoms selected from carbon, oxygen, nitrogen,
sulfur, phosphorus, silicon, boron, arsenic and selenium, wherein
the ring defined by said atoms is saturated or unsaturated,
including epoxides and thioepoxides;
[0069] A'-T' are independently selected from hydrogen, R.sub.4,
R.sub.5, R.sub.6, F, Cl, Br, I, CN, COR.sub.4, CO.sub.2R.sub.4,
OR.sub.4, SR.sub.4, NR.sub.4R.sub.5, CONR.sub.4R.sub.5,
N(.dbd.O).sub.2, NR.sub.4OR.sub.5, ONR.sub.4R.sub.5, SOR.sub.4,
SO.sub.2R.sub.4, SO.sub.3R.sub.4, SONR.sub.4R.sub.5,
S0.sub.2NR4R.sub.5, S0.sub.3NR.sub.4R.sub.5, P(R.sub.4).sub.3,
P(.dbd.O)(R.sub.4).sub.3, Si(R.sub.4).sub.3, B(R.sub.4).sub.2,
(C.dbd.X)R.sub.6 or X(C.dbd.X)R.sub.6 where X is selected from
sulfur, oxygen and nitrogen;
[0070] R.sub.4 and R.sub.5 are each independently selected from
C.sub.1-C.sub.20 alkyl (branched and/or straight chained),
C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8 cycloalkyl,
C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and/or straight chained), C.sub.2-C.sub.10 alkynyl (branched and/or
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.4, SR.sub.4, NR.sub.4R.sub.5, N(.dbd.O).sub.2,
NR.sub.4OR.sub.5, ONR.sub.4R.sub.5, SOR.sub.4, SO.sub.2R.sub.4,
SO.sub.3R.sub.4, SONR.sub.4R.sub.5, SO.sub.2NR.sub.4R.sub.5,
SO.sub.3NR.sub.4R.sub.5, P(R.sub.4).sub.3,
P(.dbd.O)(R.sub.4).sub.3, Si(R.sub.4).sub.3,
B(P.sub.4).sub.2]alkyl;
[0071] R.sub.6 is selected from R.sub.4, R.sub.5, CN, COR.sub.4,
CO.sub.2R.sub.4, OR.sub.4, SR.sub.4, NR.sub.4R.sub.5,
N(.dbd.O).sub.2, NR.sub.4OR.sub.5, ONR.sub.4R.sub.5, SOR.sub.4,
SO.sub.2R.sub.4, SO.sub.3R.sub.4, SONR.sub.4R.sub.5,
SO.sub.2NR.sub.4R.sub.5, SO.sub.3NR.sub.4R.sub.5, P(R.sub.4).sub.3,
P(.dbd.O)(R.sub.4).sub.3, Si(R.sub.4).sub.3, B(R.sub.4).sub.2;
[0072] E' and R' or H' and O' is a C.sub.2-C.sub.8 saturated or
unsaturated carbocyclic or heterocyclic ring system further
substituted by R.sub.6, including epoxides and thioepoxides;
[0073] O' connected to M' (or N') or Q' (or P'); R' connected to Q'
(or P') or S' (or T'); S' (or T') connected to A' (or B'); A' (or
B') connected to C' (or D'); E' connected to C' (or D') or F' (or
G'); H' connected to I'; I' connected to J'; J' connected to K'; K'
connected to L'; L' connected to M' (or N') are C.sub.1-C.sub.8
disubstituted (fused) saturated or unsaturated carbocyclic or
heterocyclic ring systems further substituted by R.sub.6,
(C.dbd.X)R.sub.6 and X(C.dbd.X)R.sub.6, including epoxides and
thioepoxides;
[0074] A', B' and C', D' and F', G' and M', N' and P', Q' and S',
T' are .dbd.X where X is selected from sulfur, oxygen, nitrogen,
NR.sub.4R.sub.5, (C.dbd.X)R.sub.6, X(C.dbd.X)R.sub.6, and
.dbd.CR.sub.7R.sub.8;
[0075] R.sub.7 and R.sub.8 are each independently selected from
R.sub.6, (C.dbd.X)R.sub.6 and X(C.dbd.X)R.sub.6 3
[0076] wherein:
[0077] n is 0-10 atoms selected from carbon, oxygen, nitrogen,
sulfur, phosphorus, silicon, boron, arsenic and selenium, wherein
the ring defined by said atoms is saturated or unsaturated,
including epoxides and thioepoxides;
[0078] A.sup.1-T.sup.1 are independently selected from hydrogen,
R.sub.9, R.sub.10, R.sub.11, F, Cl, Br, I, CN, OR.sub.9, SR.sub.9,
NR.sub.9R.sub.10, N(.dbd.O).sub.2, NR.sub.9OR.sub.10,
ONR.sub.9R.sub.10, SOR.sub.9, SO.sub.2R.sub.9, SO.sub.3R.sub.9,
SONR.sub.9R.sub.10, SO.sub.2NR.sub.9R.sub.10,
SO.sub.3NR.sub.9R.sub.10, P(R.sub.9).sub.3,
P(.dbd.O)(R.sub.9).sub.3, Si(R.sub.9).sub.3, B(R.sub.9).sub.2,
(C.dbd.X)R.sub.11 or X(C.dbd.X)R.sub.11 where X is selected from
sulfur, oxygen and nitrogen;
[0079] R.sub.9 and R.sub.10 are each independently selected from
C.sub.1-C.sub.20 alkyl (branched and straight chained),
C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8 cycloalkyl,
C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and straight chained), C.sub.2-C.sub.10 alkynyl (branched and
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.9, SR.sub.9, NR.sub.9R.sub.10, N(.dbd.O).sub.2,
NR.sub.9OR.sub.10, ONR.sub.9R.sub.10, SOR.sub.9, SO.sub.2R.sub.9,
SO.sub.3R.sub.9, SONR.sub.9R.sub.10, SO.sub.2NR.sub.9R.sub.10,
SO.sub.3NR.sub.9R.sub.10, P(R.sub.9).sub.3,
P(.dbd.O)(R.sub.9).sub.3, Si(R.sub.9).sub.3,
B(R.sub.9).sub.2]alkyl;
[0080] R.sub.11 is selected from R.sub.9, R.sub.10, CN, COR.sub.9,
CO.sub.2R.sub.9, OR.sub.9, SR.sub.9, NR.sub.9R.sub.10,
N(.dbd.O).sub.2, NR.sub.9OR.sub.10, ONR.sub.9R.sub.10, SOR.sub.9,
SO.sub.2R.sub.9, SO.sub.3R.sub.9, SONR.sub.9R.sub.10,
SO.sub.2NR.sub.9R.sub.10, SO.sub.3NR.sub.9R.sub.10,
P(R.sub.9).sub.3, P(.dbd.O)(R.sub.9).sub.3, Si(R.sub.9).sub.3,
B(R.sub.9).sub.2;
[0081] B.sup.1 and R.sup.1, E.sup.1 and .sup.1 and .sup.1 and
M.sup.1 are selected from a C.sub.2-C.sub.8 saturated or
unsaturated carbocyclic or heterocyclic ring system further
substituted by R.sub.11, including epoxides and thioepoxides;
[0082] A.sup.1 (or .sup.1 connected to .sup.1 (or .sup.1) or
T.sup.1 (or S.sup.1) B.sup.1 connected to .sup.1 (or .sup.1) or
C.sup.1 (or D.sup.1) E.sup.1 connected to .sup.1 or C.sup.1 (or
D.sup.1); .sup.1 connected to .sup.1 (or F.sup.1); G.sup.1 (or
H.sup.1) connected to .sup.1 (or F.sup.1) or I.sup.1 (or J.sup.1);
K.sup.1 (or L.sup.1) connected to I.sup.1 (or J.sup.1) or M.sup.1;
M.sup.1 connected to O.sup.1 (or N.sup.1); .sup.1 connected O.sup.1
(or N.sup.1) or P.sup.1 (or Q.sup.1); R.sup.1 connected P.sup.1 (or
Q.sup.1) or S.sup.1 (or T.sup.1) are C.sub.1-C.sub.8 disubstituted
(fused) saturated or unsaturated carbocyclic or heterocyclic ring
systems further substituted by R.sub.11, (C.dbd.X)R.sub.11 and
X(C.dbd.X)R.sub.11, including epoxides and thioepoxides;
[0083] A.sup.1, and , and C.sup.1, D.sup.1 and F.sup.1, and
G.sup.1, H.sup.1 and I.sup.1, J.sup.1 and K.sup.1, L.sup.1 and
N.sup.1, O.sup.1 and P.sup.1, Q.sup.1 and S.sup.1, T.sup.1 are
.dbd.X where X is selected from sulfur, oxygen, nitrogen,
NR.sub.9R.sub.10, including (C.dbd.X)R.sub.11 and
X(C.dbd.X)R.sub.11, and .dbd.CR.sub.12R.sub.13;
[0084] R.sub.12 and R.sub.13 are independently selected from
R.sub.11, (C.dbd.X)R.sub.11 and X(C.dbd.X)R.sub.11 4
[0085] wherein:
[0086] n is 0-10 atoms selected from carbon, oxygen, nitrogen,
sulfur, phosphorus, silicon, boron, arsenic and selenium, wherein
the ring defined by said atoms is saturated or unsaturated,
including epoxides and thioepoxides;
[0087] A.sup.2-X.sup.2 are independently selected from hydrogen,
R.sub.14, R.sub.15, R.sub.16, F, Cl, Br, I, CN, OR.sub.14,
SR.sub.14, NR.sub.14R.sub.15, N(.dbd.O).sub.2, NR.sub.14OR.sub.15,
ONR.sub.14R.sub.15, SOR.sub.14, SO.sub.2R.sub.14, SO.sub.3R.sub.14,
SONR.sub.14R.sub.15, SO.sub.2NR.sub.14R.sub.15,
SO.sub.3NR.sub.14R.sub.15- , P(R.sub.14).sub.3,
P(.dbd.O)(R.sub.14).sub.3, Si(R.sub.14).sub.3, B(R.sub.14),
(C.dbd.Y)R.sub.16 or Y(C.dbd.Y)R.sub.16 where Y is selected from
sulfur, oxygen and nitrogen;
[0088] R.sub.14 and R.sub.15 are each independently selected from
C.sub.1-C.sub.20 alkyl (branched and/or straight chained),
C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8 cycloalkyl,
C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and/or straight chained), C.sub.2-C.sub.10 alkynyl (branched and/or
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.14, SR.sub.14, NR.sub.14R.sub.10, N(.dbd.O).sub.2,
NR.sub.14OR.sub.15, ONR.sub.14R.sub.15, SOR.sub.14,
SO.sub.2R.sub.14, SO.sub.3R.sub.14, SONR.sub.14R.sub.15,
SO.sub.2NR.sub.14R.sub.15, SO.sub.3NR.sub.14R.sub.15,
P(R.sub.14).sub.3, P(.dbd.O)(R.sub.14).sub.3, Si(R.sub.14).sub.3,
B(R.sub.14).sub.2]alkyl;
[0089] R.sub.16 is selected from R.sub.14, R.sub.15, CN,
COR.sub.14, CO.sub.2R.sub.15, OR.sub.14, SR.sub.14,
NR.sub.14R.sub.15, N(.dbd.O).sub.2, NR.sub.14OR.sub.15,
ONR.sub.14R.sub.15, SOR.sub.14, SO.sub.2R.sub.14, SO.sub.3R.sub.14,
SONR.sub.14R.sub.15, SO.sub.2NR.sub.14R.sub.15,
SO.sub.3NR.sub.14R.sub.15, P(R.sub.14).sub.3,
P(.dbd.O)(R.sub.14).sub.3, Si(R.sub.14).sub.3,
B(R.sub.14).sub.2;
[0090] E.sup.2 and V.sup.2, H.sup.2 and S.sup.2, and I.sup.2 and
P.sup.2 are C.sub.2-C.sub.8 saturated or unsaturated carbocyclic or
heterocyclic ring system further substituted by R.sub.16, including
epoxides and thioepoxides;
[0091] A.sup.2 (or B.sup.2) connected to C.sup.2 (or D.sup.2) or
W.sup.2 (or X.sup.2); E.sup.2 connected to C.sup.2 (or D.sup.2) or
F.sup.2 (or G.sup.2); H.sup.2 connected to F.sup.2 (or G.sup.2) or
I.sup.2; I.sup.2 connected to J.sup.2 (or K.sup.2); L.sup.2 (or
M.sup.2) connected to J.sup.2 (or K.sup.2) or N.sup.2 (or O.sup.2);
R.sup.2 (or Q.sup.2) connected to P.sup.2 or S.sup.2; V.sup.2
connected to U.sup.2 (or T.sup.2) or W.sup.2 (or X.sup.2) are
C.sub.1-C.sub.8 disubstituted (fused) saturated or unsaturated
carbocyclic or heterocyclic ring systems further substituted by
R.sub.16, (C.dbd.Y)R.sub.16 and Y(C.dbd.Y)R.sub.16, including
epoxides and thioepoxides;
[0092] A.sup.2, B.sup.2; C.sup.2, D.sup.2; F.sup.2, G.sup.2;
J.sup.2, K.sup.2; L.sup.2, M.sup.2; N.sup.2, O.sup.2; Q.sup.2,
R.sup.2; U.sup.2, T.sup.2 and X.sup.2, W.sup.2 are .dbd.Y where Y
is selected from sulfur, oxygen, nitrogen, NR.sub.14R.sub.15 and
.dbd.CR.sub.17R.sub.18;
[0093] R.sub.17 and R.sub.18 are independently selected from
R.sub.16, (C.dbd.Y)R.sub.16 and Y(C.dbd.Y)R.sub.16 5
[0094] wherein:
[0095] n is 0-10 atoms selected from carbon, oxygen, nitrogen,
sulfur, phosphorus, silicon, boron, arsenic and selenium, wherein
the ring defined by said atoms is saturated or unsaturated,
including epoxides and thioepoxides;
[0096] A.sup.3-Z.sup.3 are independently selected from hydrogen,
R.sub.19, R.sub.20, R.sub.21, F, Cl, Br, I, CN, OR.sub.19,
SR.sub.19, NR.sub.19R.sub.20, N(.dbd.O).sub.2, NR.sub.19OR.sub.20,
ONR.sub.19R.sub.20, SOR.sub.19, SO.sub.2R.sub.19, SO.sub.3R.sub.19,
SONR.sub.19R.sub.20, SO.sub.2NR.sub.19R.sub.20,
SO.sub.3NR.sub.19R.sub.20- , P(R,.sub.9).sub.3,
P(O)(R.sub.19).sub.3, Si(R.sub.19).sub.3, B(R.sub.19).sub.2,
(C.dbd..O slashed.)R.sub.21 or .O slashed.(C.dbd..O
slashed.)R.sub.21 where .O slashed. is sullur, oxygen and
nitrogen;
[0097] R.sub.19 and R.sub.20 are each independently selected from
C.sub.1-C.sub.20 alkyl (branched and/or straight chained),
C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8 cycloalkyl,
C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and/or straight chained), C.sub.2-C.sub.10 alkynyl (branched and/or
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.19, SR.sub.19, NR.sub.19R.sub.20, N(.dbd.O).sub.2,
NR.sub.19OR.sub.20, ONR.sub.19R.sub.20, SOR.sub.19,
SO.sub.2R.sub.19, SO.sub.3R.sub.19, SONR.sub.19R.sub.20,
SO.sub.2NR.sub.19R.sub.20, SO.sub.3NR.sub.19R.sub.20,
P(R.sub.19).sub.3, P(.dbd.O)(R.sub.19).sub.3, Si(R.sub.19).sub.3,
B(R.sub.19).sub.2]alkyl;
[0098] R.sub.21 is selected from R.sub.19, R.sub.20, CN,
COR.sub.19, CO.sub.2R.sub.19, OR.sub.19, SR.sub.19,
NR.sub.19R.sub.20, N(.dbd.O).sub.2, NR.sub.19OR.sub.20,
ONR.sub.19R.sub.20, SOR.sub.19, SO.sub.2R.sub.19, SO.sub.3R.sub.19,
SONR.sub.19R.sub.20, SO.sub.2NR.sub.19R.sub.20,
SO.sub.3NR.sub.19R.sub.20, P(R.sub.19).sub.3,
P(.dbd.O)(R.sub.19).sub.3, Si(R.sub.19).sub.3,
B(R.sub.19).sub.2;
[0099] D.sup.3 connected to X.sup.3 is a C.sub.2-C.sub.8 saturated
or unsaturated carbocyclic or heterocyclic ring system further
substituted by R.sub.21, including epoxides and thioepoxides;
[0100] A.sup.3 (or .sup.3) connected to B.sup.3 (or C.sup.3) or
E.sup.3 (or X); D.sup.3 connected to B.sup.3 (or C.sup.3) or
E.sup.3 (or F.sup.3); G.sup.3 (or H.sup.3) connected to E.sup.3 (or
F.sup.3) or I.sup.3 (or J.sup.3); L.sup.3 (or K.sup.3) connected to
I.sup.3 (or J.sup.3) or M.sup.3 (or N.sup.3); O.sup.3 (or .sup.3)
connected to N.sup.3 (or M.sup.3) or P.sup.3 (or Q.sup.3). S.sup.3
(or R.sup.3) connected to Q.sup.3 (or P.sup.3) or U.sup.3 (or
T.sup.3). W.sup.3 (or V.sup.3) connected to U.sup.3 (or T.sup.3) or
X.sup.3; X.sup.3 connected to Y.sup.3 (or Z.sup.3) are
C.sub.1-C.sub.8 disubstituted (fused) saturated or unsaturated
carbocyclic or heterocyclic ring systems further substituted by
R.sub.21, (C.dbd..O slashed.)R.sub.21 and .O slashed. (C.dbd..O
slashed.)R.sub.21, including epoxides and thioepoxides;
[0101] A.sup.3, .sup.3; B.sup.3, C.sup.3; E.sup.3, F.sup.3;
G.sup.3, H.sup.3; I.sup.3, J.sup.3; K.sup.3, L.sup.3; M.sup.3,
N.sup.3; O.sup.3, .sup.3; Q.sup.3, P.sup.3, S.sup.3, R.sup.3,
U.sup.3, T.sup.3, W.sup.3, V.sup.3, and Z.sup.3, Y.sup.3 are
.dbd..O slashed. where .O slashed. is selected from sulfur, oxygen,
nitrogen, NR.sub.19R.sub.20, and .dbd.CR.sub.22R.sub.23; and
[0102] R.sub.22 and R.sub.23 are selected from R.sub.21, (C.dbd..O
slashed.)R.sub.21 and .O slashed.(C.dbd..O slashed.)R.sub.21;
[0103] and which chemical agent or derivative or chemical analogue
thereof is capable of modulating PKC activity, PKC-gene expression
or PKC enzyme turnover and wherein said chemical agent or its
derivatives or chemical analogues is administered for a time and
under conditions sufficient to ameliorate one or more symptoms
associated with a PKC-related condition or disorder.
[0104] Especially preferred chemical agents or derivatives or
chemical analogues thereof are represented by the general formula
(VI): 6
[0105] wherein:
[0106] R.sub.24, R.sub.25 and R.sub.26 are independently selected
from hydrogen, R.sub.27, R.sub.28, F, Cl, Br, I, CN, OR.sub.27,
SR.sub.27, NR.sub.27R.sub.28, N(.dbd.O).sub.2, NR.sub.27OR.sub.28,
ONR.sub.27R.sub.28, SOR.sub.27, SO.sub.2R.sub.27, SO.sub.3R.sub.27,
SONR.sub.27R.sub.28, SO.sub.2NR.sub.27R.sub.28,
SO.sub.3NR.sub.27R.sub.28- , P(R.sub.27).sub.3,
P(.dbd.O)(R.sub.27).sub.3, Si(R.sub.27).sub.3, B(R.sub.27).sub.2,
(C.dbd.X)R.sub.29 or X(C.dbd.X)R.sub.29 where X is selected from
sulfur, oxygen and nitrogen;
[0107] R.sub.27 and R.sub.28 are each independently selected from
C.sub.1-C.sub.20 alkyl (branched and/or straight chained),
C.sub.1-C.sub.20 arylalkyl, C.sub.3-C.sub.8 cycloalkyl,
C.sub.6-C.sub.14 aryl, C.sub.1-C.sub.14 heteroaryl,
C.sub.1-C.sub.14 heterocycle, C.sub.2-C.sub.10 alkenyl (branched
and/or straight chained), C.sub.2-C.sub.10 alkynyl (branched and/or
straight chained), C.sub.1-C.sub.10 heteroarylalkyl,
C.sub.1-C.sub.10 alkoxyalkyl, C.sub.1-C.sub.10 haloalkyl,
dihaloalkyl, trihaloalkyl, haloalkoxy, C.sub.1-C.sub.10 [CN,
OR.sub.27, SR.sub.27, NR.sub.27R.sub.28, N(.dbd.O).sub.2,
NR.sub.27OR.sub.28, ONR.sub.27R.sub.28, SOR.sub.27,
SO.sub.2R.sub.27, SO.sub.3R.sub.27, SONR.sub.27R.sub.28,
SO.sub.2NR.sub.27R.sub.28, SO.sub.3NR.sub.27R.sub.28,
P(R.sub.27).sub.3, P(.dbd.O)(R.sub.27).sub.3, Si(R.sub.27).sub.3,
B(R.sub.27).sub.2]alkyl;
[0108] R.sub.29 is selected from R.sub.27, R.sub.28, CN,
COR.sub.27, CO.sub.2R.sub.27, OR.sub.27, SR.sub.27,
NR.sub.27R.sub.28, N(.dbd.O).sub.2, NR.sub.27OR.sub.28,
ONR.sub.27R.sub.28, SOR.sub.27, S0.sub.2R.sub.27, SO.sub.3R.sub.27,
SONR.sub.27R.sub.28, SO.sub.2NR.sub.27R.sub.28,
SO.sub.3NR.sub.27R.sub.28, P(R.sub.27).sub.3,
P(.dbd.O)(R.sub.27).sub.3, Si(R.sub.27).sub.3,
B(R.sub.27).sub.2.
[0109] In a preferred embodiment, R.sub.24 is hydrogen, OAcetyl or
OH.
[0110] In another preferred embodiment, R.sub.25 and R.sub.26 are
OH.
[0111] As used herein, the term "alkyl" refers to linear or
branched chains. The term "haloalkyl" refers to an alkyl group
substituted by at least one halogen. Similarly, the term
"haloalkoxy" refers to an alkoxy group substituted by at least one
halogen. As used herein the term "halogen" refers to fluorine,
chlorine, bromine and iodine.
[0112] As used herein the term "aryl" refers to aromatic
carbocyclic ring systems such as phenyl or naphthyl, anthracenyl,
especially phenyl; Suitably, aryl is C.sub.6-C.sub.14 with mono,
di- and tri- substitution containing F, Cl, Br, I, NO.sub.2,
CF.sub.3, CN, OR.sub.1, COR.sub.1, CO.sub.2R.sub.1, NHR.sub.1,
NR.sub.1R.sub.2, NR.sub.1OR.sub.2, ONR.sub.1R.sub.2, SOR.sub.1,
SO.sub.2R.sub.1, SO.sub.3R.sub.1, SONR.sub.1R.sub.2,
SO.sub.2NR.sub.1R.sub.2, SO.sub.3NR.sub.1R.sub.2, P(R.sub.1).sub.3,
P(.dbd.O)(R.sub.1).sub.3, Si(R.sub.1).sub.3, B(R.sub.1).sub.2,
wherein R.sub.1 and R.sub.2 are defined above
[0113] As used herein the terms "heterocycle", "heterocyclic",
"heterocyclic systems" and the like refer to a saturated,
unsaturated, or aromatic carbocyclic group having a single ring,
multiple fused rings (for example, bicyclic, tricyclic, or other
similar bridged ring systems or substituents), or multiple
condensed rings, and having at least one heteroatom such as
nitrogen, oxygen, or sulfur within at least one of the rings. This
term also includes "heteroaryl" which refers to a heterocycle in
which at least one ring is aromatic. Any heterocyclic or heteroaryl
group can be unsubstituted or optionally substituted with one or
more groups, as defined above. Further, bi- or tricyclic heteroaryl
moieties may comprise at least one ring, which is either
completely, or partially, saturated. Suitable heteroaryl moieties
include, but are not limited to oxazolyl, thiazaoyl, thienyl,
furyl, 1-isobenzofuranyl, 3H-pyrrolyl, 2H-pyrrolyl, N-pyrrolyl,
imidazolyl, pyrazolyl, isothiazolyl, isooxazolyl, pyridyl,
pyrazinyl, pyrimidinyl, pyradazinyl, indolizinyl, isoindolyl,
indoyl, indolyl, purinyl, phthalazinyl, 1,2,3-triazolyl,
1,2,4-triazolyl, 1,2,3-oxadiazoyl, 1,2,4-oxadiazolyl,
1,2,5-oxadiazolyl, 1,3,4-oxadiazolyl, 1,2,3,4-oxatriazolyl,
1,2,3,5-oxatriazolyl, 1,3,5-triazinyl, 1,2,4-triazinyl,
1,2,3-triazinyl, azepinyl, oxepinyl, thiepinyl, benzofuranyl,
isobenzofuranyl, thionaphthenyl, isothionaphthenyl, indoleninyl,
2-isobenzazolyl, 1,5-pyrindinyl, pyrano[3,4-b]pyrrolyl,
isoindazolyl, indoxazinyl, benzoxazolyl, anthranilyl, quinolinyl,
isoquinolinyl, cinnolinyl, quinazolinyl, naphthyridinyl,
pyrido[3,4-b]pyridinyl, and pyrido[3,2-b]pyridinyl,
pyrido[4,3-b]pyridinyl.
[0114] Preferred PKC-related conditions include alcoholism,
Alzheimer's disease, asthma, atherosclosis, atopic dermatitis,
autoimmune disease, bipolar-disorder, blood disorder, cardiac
hypertrophy, depression, diabetes, hypertension, hyperplastic
dermatosis, multiple sclerosis, myocardial ischemia,
osteoarthritis, psoriasis, rheumatoid arthritis, transplantation
and a latent virus. The instant chemical agents are also useful in
promoting gene activation and promotion.
[0115] The subject chemical agents are also useful in modulating
and more particularly promoting or activating gene expression or
the expression or operation of a promoter or other regulatory
sequence. This aspect is useful for gene therapy, gene augmentation
or gene replacement therapy or the provision of expression products
(e.g. peptides, polypeptides, proteins, antisense RNA, sense RNA)
by endogenous genes or exogenously introduced DNA or RNA.
[0116] The subject compounds are also useful in activating latent
viruses.
[0117] The term "viruses" is used in its broadest sense to include
viruses of the families adenoviruses, papovaviruses, herpesviruses:
simplex, varicella-zoster, Epstein-Barr, CMV, pox viruses:
smallpox, vaccinia, hepatitis B, rhinoviruses, hepatitis A,
poliovirus, rubellavirus, hepatitis C, arboviruses, rabiesvirus,
influenzaviruses A and B, measlesvirus, mumpsvirus, HIV, HTLV I and
II.
[0118] Particularly preferred viruses are HIV, EBV and CMV.
[0119] Accordingly, another aspect of the present invention provide
a method for the treatment or prophylaxis of a PKC-related
condition or disorder in a subject, said method comprising the
administration to said subject of a symptom-ameliorating effective
amount of a macrocyclic diterpene, or a chemical fraction
comprising same from a plant of the family Euphorbiaceae or a
derivative or chemical analogue of said macrocyclic diterpene
having the structures as defined above wherein said macrocyclic
diterpene or its derivative or chemical analogue modulates PKC
activity, synthesis or enzyme turnover, said administration being
for a time and under conditions sufficient to ameliorate one or
more symptoms of associated with a PKC-related condition or
disorder.
[0120] This aspect of the present invention is particularly useful
in the treatment of latent virus infection. The term "latent virus"
includes reference to a virus or more particularly a virus genome
or part thereof which has integrated into the genome of a cell.
When in the latent state, it is more difficult for a host's immune
system to recognize a virus as a foreign body. In accordance with
the present invention, it is proposed that the subject chemical
agents are capable of activating a latent virus thereby causing the
virus to undergo replication and at least partial assembly. As a
result, a mechanism within the host or within the cells of the host
is then induced to assist in the eradication of the virus. The
present invention extends to both the direct effect of the chemical
agent on the virus as well as promoting the immune system to direct
same against the virus.
[0121] Accordingly, another aspect of the present invention
contemplates a method for the treatment or prophylaxis of a
PKC-related condition or disorder relating to a latent virus in a
subject, said method comprising the administration to said subject
of a virus-activating effective amount of a macrocyclic diterpene,
or a chemical fraction comprising same from a plant of the family
Euphorbiaceae or a derivative or chemical analogue of said
macrocyclic diterpene having the structures as defined above
wherein said macrocyclic diterpene or its derivative or chemical
analogue modulates PKC activity, synthesis or enzyme turnover, said
administration being for a time and under conditions sufficient to
activate said virus and to combination therapies with antiviral
agents.
[0122] The activated virus is then destroyed or removed by the
host's own immune system and/or by the effects of the agents
themselves.
[0123] In a preferred embodiment, the method further comprises the
simultaneous, sequential or separate administration of an ancillary
agent that destroys or attenuates a replicating virus, in
combination with the macrocyclic diterpene or chemical fraction.
Thus, the invention contemplates combination therapies in which the
macrocyclic diterpene or chemical fraction activates a latent virus
to thereby cause the virus to undergo replication and the ancillary
agent eradicates the replicating virus.
[0124] Accordingly, in another aspect, the invention encompasses a
method for the treatment or prophylaxis of a PKC-related condition
or disorder relating to a latent virus in a subject, said method
comprising simultaneously, sequentially or separately
administrating to said subject a virus-activating effective amount
of a macrocyclic diterpene, or a chemical fraction comprising same
from a plant of the family Euphorbiaceae or a derivative or
chemical analogue of said macrocyclic diterpene having the
structures as defined above wherein said macrocyclic diterpene or
its derivative or chemical analogue modulates PKC activity,
synthesis or enzyme turnover, together with a virus-destroying or
-attenuating effective amount of an ancillary agent which destroys
or attenuates a replicating form of said virus, said administration
being for a time and under conditions sufficient for said
macrocyclic diterpene or chemical fraction to activate said virus
to thereby cause the virus to undergo replication and for said
ancillary agent to destroy or attenuate said replicating virus.
[0125] In one embodiment, the latent virus is HIV. A range of HIV
specific agents may be used for the destruction or attenuation of
this virus, including, for example, those described in Matsuhita et
al., 2000. Preferred HIV specific ancillary agents include, for
example, nucleoside analogues such as combivir, epivir, hivid,
retrovir, videx, zerat and zygen, non-nucleoside agents such as
rescriptor, sustiva and viramune, adjunctive anti-retrovirals such
as hydrea and droxa, and protease inhibitors such as agenerase,
fortovase, crixivan, invirase, norvir and virasept.
[0126] In another embodiment, the latent virus is EBV. Preferred
EBV specific ancillary agents are selected from ganciclovir (GVC)
or 3'-azido-3'deoxythymidine (AZT).
[0127] In yet another embodiment, the latent virus is CMV. A
preferred CMV specific ancillary agent is cidofovir.
[0128] Particularly useful compounds include
5,8,9,10,14-pentaacetoxy-3-be- nzoyloxy- 15-hydroxypepluane
(pepluane), derivatives of said pepluane, jatrophanes of
Conformation II including 2,3,5,7,15-pentaacetoxy-9-nicoti-
noyloxy-14-oxojatropha-6(17),11E-diene kjatrophane 1), derivatives
of said jatrophane
1,2,5,7,8,9,14-hexaacetoxy-3-benzoyloxy-15-hydroxy-jatropha-6(-
17),11E-diene (jatrophane 2), derivatives of said jatrophane 2,
2,5,14-triacetoxy-3-benzoyloxy-8,15-dihydroxy-7-isobutyroyloxy-9-nicotino-
yloxy-jatropha-6(17),11E-diene (jatrophane 3), derivatives of said
jatrophane 3,
2,5,9,14-tetraacetoxy-3-benzoyloxy-8,15-dihydroxy-7-isobuty-
royloxyjatropha-6(17),11E-diene) (jatrophane 4), derivatives of
said jatrophane 4,
2,5,7,14-tetraacetoxy-3-benzoyloxy-8,15-dihydroxy-9-nicotin-
oyloxyjatropha-6(17), 11E-diene (jatrophane 5), derivatives of said
jatrophane 5,
2,5,7,9,14-pentaacetoxy-3-benzoyloxy-8,15-dihydroxyjatropha- -6(
17), 11E-diene jatrophane 6), derivatives of said jatrophane 6, or
pharmaceutically acceptable salts of these.
[0129] Even more particularly preferred compounds are angeloyl
substituted ingenanes or derivatives thereof such as
ingenol-3-angelate, 20-hydroxy-ingenol-3-angelate,
20-O-acetyl-ingenol-3-angelate, or derivatives of said angelates,
or pharmaceutically acceptable salts of these.
[0130] Still a further aspect of the present invention contemplates
a method of assessing the suitability of a chemical agent from
Euphorbiaceae for the practice of the present invention. Numerical
values are assigned to chemical agents including fractions
comprising the chemical agents as set forth, for example, in Table
A:
2 TABLE A Feature Value An ability to modulate PKC activity or
effect +1 An ability to induce bipolar dendritic activity +1 An
ability to displace phorbol dibutyrate from +1 binding to PKC An
ability to induce respiratory burst in leucocytes +1 An ability to
stimulate phagocytosis in peripheral +1 blood mononuclear cells
Derived from a member of the Euphorbiaeae family +1 Derived from E.
peplus +3 Water extractible from the sap of Euphorbia sp. +2 An
ability to activate latent virus +4 A lower tumor promotion
activity than TPA/PMA +2
[0131] The value for each feature is referred to as the Index Value
(I.sub.V).
[0132] The sum of I.sub.V, i.e. .SIGMA.I.sub.V, provides a potency
of agent (P.sub.A) value and this enables an analytical approach to
screening and selecting compounds from Euphorbiaceae useful in the
practice of the present invention.
[0133] In one example, 20-acetyl-ingenol-3 angelate exhibits a
P.sub.A=.SIGMA.I.sub.V=15.
[0134] Accordingly, another aspect of the present invention
contemplates a method for the treatment or prophylaxis of a
PKC-related condition in a subject, said method comprising
administration to said subject of a symptom-ameliorating effective
amount of a macrocyclic diterpene obtainable from a Euphorbiaceae
plant or its botanical or horticultural relative, said macrocyclic
diterpene being selected from an ingenane, pepluane or jatrophane,
or a derivative or chemical analogue thereof, having the structure
represented by any one of the general formulae (I)-(V) as defined
above and wherein said chemical agent exhibits a potency of agent
(P.sub.A) of >10, wherein the P.sub.A=.SIGMA.I.sub.V where
I.sub.V is a numerical value associated with a particular feature
as listed below:
3 Feature Value An ability to modulate PKC activity or effect +1 An
ability to induce bipolar dendritic activity +1 An ability to
displace phorbol dibutyrate from +1 binding to PKC An ability to
induce respiratory burst in leucocytes +1 An ability to stimulate
phagocytosis in peripheral +1 blood mononuclear cells Derived from
a member of the Euphorbiaceae family +1 Derived from E. peplus +3
Water extractible from the sap of Euphorbia sp. +2 An ability to
activate latent virus +4 A lower tumor promotion activity than
TPA/PMA +2
[0135] or pharmaceutically acceptable salts of these, said chemical
agent being administered for a time and under conditions sufficient
to ameliorate at least one symptom caused by or associated with the
biological entity.
[0136] Preferred compounds are selected from the list
comprising:
[0137] 5,8,9,10,14-pentaacetoxy-3-benzoyloxy-15-hydroxypepluane
(pepluane);
[0138]
2,3,5,7,15-pentaacetoxy-9-nicotinoyloxy-14-oxojatropha-6(17),11E-di-
ene (jatrophane 1);
[0139]
2,5,7,8,9,14-hexaacetoxy-3-benzoyloxy-15-hydroxy-jatropha-6(17),11E-
-diene (jatrophane 2);
[0140]
2,5,14-triacetoxy-3-benzoyloxy-8,15-dihydroxy-7-isobutyroyloxy-9-ni-
cotinoyloxy-jatropha-6(17),11E-diene (jatrophane 3);
[0141]
2,5,9,14-tetraacetoxy-3-benzoyloxy-8,15-dihydroxy-7-isobutyroyloxy
jatropha-6(17),11E-diene) (jatrophane 4);
[0142]
2,5,7,14-tetraacetoxy-3-benzoyloxy-8,15-dihydroxy-9-nicotinoyloxy-j-
atropha-6(17),11E-diene (jatrophane 5);
[0143]
2,5,7,9,14-pentaacetoxy-3-benzoyloxy-8,15-dihydroxyjatropha-6(17),1-
1E-diene (jatrophane 6);
[0144] 20-O-acetyl-ingenol-3-angelate, derivatives of
20-O-acetyl-ingenol-3-angelate.
[0145] 20-hydroxy-ingelol-3-angelate, derivatives of
20-hydroxy-ingenol-3-angelate; and
[0146] ingenol-3-angelate, derivatives of ingenol-3-angelate.
[0147] Preferably, the biological entity is a microorganism, virus,
yeast, fungus, insect, arachnid or Plasmodium sp.
[0148] Reference herein to a subject includes a human, primate,
livestock animal (e.g. sheep, cow, horse, pig, goat, donkey),
laboratory test animal (e.g. mouse, rat, guinea pig, hamster),
companion animal (e.g. dog, cat) or avian species such as poultry
birds (e.g. chicken, ducks, turkeys, geese) or game birds (e.g.
arid ducks, pheasants).
[0149] The preferred subject is a human or primate or laboratory
test animal.
[0150] The most preferred subject is a human.
[0151] The ability to assign numerical values to certain
characteristics enables data processing means to assess the likely
usefulness of a particular compound or group of compounds forming a
chemical agent.
[0152] The assessment of the suitability of a compound or group of
compounds for the practice of the present invention is suitably
facilitated with the assistance of a computer programmed with
software, which inter alia adds index values (I.sub.V) for at least
two features associated with the compound(s) to provide a potency
value (P.sub.A) corresponding to the effectiveness of the
compound(s) for treating or preventing infection or colonization or
presence of a biological entity in a subject. The compound features
can be selected from:
[0153] (a) the ability to modulate PKC activity or effect;
[0154] (b) the ability to induce bipolar dendritic activity;
[0155] (c) the ability to be derived from a member of the
Euphorbiaceae family;
[0156] (d) the ability to be derived from E. peplus;
[0157] (e) the ability to be water extractable from the sap of a
Euphorbia species;
[0158] (f) the ability to activate latent virus; or
[0159] (g) less tumor promoting capacity than TPA or MPA.
[0160] Accordingly, in accordance with the present invention, index
values for such features are stored in a machine-readable storage
medium, which is capable of processing the data to provide a
potency value for a compound or group of compounds of interest.
[0161] Thus, in another aspect, the invention contemplates a
computer program product for assessing the likely usefulness of a
candidate compound or group of compounds for treating or preventing
a PKC-related condition or disorder in a subject, said product
comprising:
[0162] (1) code that receives as input index values for at least
two features associated with said compound(s), wherein said
features are selected from:
[0163] (a) the ability to modulate PKC activity or effect;
[0164] (b) the ability to induce bipolar dendritic activity;
[0165] (c) the ability to be derived from a member of the
Euphorbiaceae family;
[0166] (d) the ability to be derived from E. peplus;
[0167] (e) the ability to be water extractable from the sap of a
Euphorbia species;
[0168] (f) the ability to activate latent virus; or
[0169] (g) less tumor promoting capacity than TPA or MPA;
[0170] (2) code that adds said index values to provide a sum
corresponding to a potency value for said compound(s); and
[0171] (3) a computer readable medium that stores the codes.
[0172] In a preferred embodiment, the computer program product
comprises code that assigns an index value for each feature of a
compound or group of compounds. In an especially preferred
embodiment, index values are assigned as set forth in Table A
above.
[0173] In a related aspect, the invention extends to a computer for
assessing the likely usefulness of a candidate compound or group of
compounds for treating or preventing a PKC-related condition or
disorder in a subject, wherein said computer comprises:
[0174] (1) a machine-readable data storage medium comprising a data
storage material encoded with machine-readable data, wherein said
machine-readable data comprise index values for at least two
features associated with said compound(s), wherein said features
are selected from:
[0175] (a) the ability to modulate PKC activity or effect;
[0176] (b) the ability to induce bipolar dendritic activity;
[0177] (c) the ability to be derived from a member of the
Euphorbiaceae family;
[0178] (d) the ability to be derived from E. peplus;
[0179] (e) the ability to be water extractable from the sap of a
Euphorbia species;
[0180] (f) the ability to activate latent virus; or
[0181] (g) less tumor promoting capacity than TPA or MPA;
[0182] (2) a working memory for storing instructions for processing
said machine-readable data;
[0183] (3) a central-processing unit coupled to said working memory
and to said machine-readable data storage medium, for processing
said machine readable data to provide a sum of said index values
corresponding to a potency value for said compound(s); and
[0184] (4) an output hardware coupled to said central processing
unit, for receiving said potency value.
[0185] A version of these embodiments is presented in FIG. 8, which
shows a system 10 including a computer 11 comprising a central
processing unit ("CPU") 20, a working memory 22 which may be, e.g.
RAM (random-access memory) or "core" memory, mass storage memory 24
(such as one or more disk drives or CD-ROM drives), one or more
cathode-ray tube ("CRT") display terminals 26, one or more
keyboards 28, one or more input lines 30, and one or more output
lines 40, all of which are interconnected by a conventional
bidirectional system bus 50.
[0186] Input hardware 36, coupled to computer 11 by input lines 30,
may be implemented in a variety of ways. For example,
machine-readable data of this invention may be inputted via the use
of a modem or modems 32 connected by a telephone line or dedicated
data line 34. Alternatively or additionally, the input hardware 36
may comprise CD. Alternatively, ROM drives or disk drives 24 in
conjunction with display terminal 26, keyboard 28 may also be used
as an input device.
[0187] Output hardware 46, coupled to computer 11 by output lines
40, may similarly be implemented by conventional devices. By way of
example, output hardware 46 may include CRT display terminal 26 for
displaying a synthetic polynucleotide sequence or a synthetic
polypeptide sequence as described herein. Output hardware might
also include a printer 42, so that hard copy output may be
produced, or a disk drive 24, to store system output for later
use.
[0188] In operation, CPU 20 coordinates the use of the various
input and output devices 36, 46 coordinates data accesses from mass
storage 24 and accesses to and from working memory 22, and
determines the sequence of data processing steps. A number of
programs may be used to process the machine readable data of this
invention. Exemplary programs may use for example the following
steps:
[0189] (1) inputting input index values for at least two features
associated with said compound(s), wherein said features are
selected from:
[0190] (a) the ability to modulate PKC activity or effect;
[0191] (b) the ability to induce bipolar dendritic activity;
[0192] (c) the ability to be derived from a member of the
Euphorbiaceae family;
[0193] (d) the ability to be derived from E. peplus;
[0194] (e) the ability to be water extractable from the sap of a
Euphorbia species;
[0195] (f) the ability to activate latent virus; or
[0196] (g) less tumor promoting capacity than TPA or MPA; and
[0197] (2) adding the index values for said features to provide a
potency value for said compound(s); and (3) outputting said potency
value.
[0198] FIG. 9 shows a cross section of a magnetic data storage
medium 100 which can be encoded with machine readable data, or set
of instructions, for designing a synthetic molecule of the
invention, which can be carried out by a system such as system 10
of FIG. 10. Medium 100 can be a conventional floppy diskette or
hard disk, having a suitable substrate 101, which may be
conventional, and a suitable coating 102, which may be
conventional, on one or both sides, containing magnetic domains
(not visible) whose polarity or orientation can be altered
magnetically. Medium 100 may also have an opening (not shown) for
receiving the spindle of a disk drive or other data storage device
24. The magnetic domains of coating 102 of medium 100 are polarized
or oriented so as to encode in manner which may be conventional,
machine readable data such as that described herein, for execution
by a system such as system 10 of FIG. 8.
[0199] FIG. 10 shows a cross section of an optically readable data
storage medium 110 which also can be encoded with such a
machine-readable data, or set of instructions, for designing a
synthetic molecule of the invention, which can be carried out by a
system such as system 10 of FIG. 8. Medium 110 can be a
conventional compact disk read only memory (CD-ROM) or a rewritable
medium such as a magneto-optical disk, which is optically readable
and magneto-optically writable. Medium 100 preferably has a
suitable substrate 111, which may be conventional, and a suitable
coating 112, which may be conventional, usually of one side of
substrate 111.
[0200] In the case of CD-ROM, as is well known, coating 112 is
reflective and is impressed with a plurality of pits 113 to encode
the machine-readable data. The arrangement of pits is read by
reflecting laser light off the surface of coating 112. A protective
coating 114, which preferably is substantially transparent, is
provided on top of coating 112.
[0201] In the case of a magneto-optical disk, as is well known,
coating 112 has no pits 113, but has a plurality of magnetic
domains whose polarity or orientation can be changed magnetically
when heated above a certain temperature, as by a laser (not shown).
The orientation of the domains can be read by measuring the
polarisation of laser light reflected from coating 112. The
arrangement of the domains encodes the data as described above.
[0202] The present invention further extends to pharmaceutical
compositions useful in treating a PKC-related disorder. In this
regard, the chemical agents of the invention can be used as actives
for the treatment or prophylaxis of a condition associated with the
presence of a PKC-related disorder in a subject. The chemical
agents can be administered to a patient either by themselves, or in
pharmaceutical compositions where they are mixed with a suitable
pharmaceutically acceptable carrier.
[0203] Accordingly, the invention also provides a composition for
treatment and/or prophylaxis of a PKC-related condition or disorder
in a subject, comprising one or more chemical agents of the
invention, together with a pharmaceutically acceptable carrier
and/or diluent.
[0204] Depending on the specific conditions being treated, chemical
agents may be formulated and administered systemically or locally.
Techniques for formulation and administration may be found in
"Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton,
Pa., latest edition. Suitable routes may, for example, include
oral, rectal, transmucosal, or intestinal administration;
parenteral delivery, including intramuscular, subcutaneous,
intramedullary injections, as well as intrathecal, direct
intraventricular, intravenous, intraperitoneal, intranasal, or
intraocular injections. For injection, the chemical agents of the
invention may be formulated in aqueous solutions, preferably in
physiologically compatible buffers such as Hanks' solution,
Ringer's solution, or physiological saline buffer. For transmucosal
administration, penetrants appropriate to the barrier to be
permeated are used in the formulation. Such penetrants are
generally known in the art. Intra-muscular and subcutaneous
injection is appropriate, for example, for administration of
immunomodulatory compositions and vaccines.
[0205] The chemical agents can be formulated readily using
pharmaceutically acceptable carriers well known in the art into
dosages suitable for oral administration. Such carriers enable the
compounds of the invention to be formulated in dosage forms such as
tablets, pills, capsules, liquids, gels, syrups, slurries,
suspensions and the like, for oral ingestion by a patient to be
treated. These carriers may be selected from sugars, starches,
cellulose and its derivatives, malt, gelatine, talc, calcium
sulphate, vegetable oils, synthetic oils, polyols, alginic acid,
phosphate buffered solutions, emulsifiers, isotonic saline, and
pyrogen-free water.
[0206] Pharmaceutical compositions suitable for use in the present
invention include compositions wherein the active ingredients are
contained in an effective amount to achieve their intended purpose.
The dose of agent administered to a patient should be sufficient to
effect a beneficial response in the patient over time such as a
reduction in the symptoms associated with the presence of a
biological entity or part thereof or toxin or venom therefrom or a
genetic event caused thereby in a subject. The quantity of the
agent(s) to be administered may depend on the subject to be treated
inclusive of the age, sex, weight and general health condition
thereof. In this regard, precise amounts of the agent(s) for
administration will depend on the judgement of the practitioner. In
determining the effective amount of the chemical agent to be
administered in the treatment or prophylaxis of a condition
associated with the biological entity, the physician may evaluate
tissue or fluid levels of the biological entity, and progression of
the disorder. In any event, those of skill in the art may readily
determine suitable dosages of the chemical agents of the
invention.
[0207] Pharmaceutical formulations for parenteral administration
include aqueous solutions of the active compounds in water-soluble
form. Additionally, suspensions of the active compounds may be
prepared as appropriate oily injection suspensions. Suitable
lipophilic solvents or vehicles include fatty oils such as sesame
oil, or synthetic fatty acid esters, such as ethyl oleate or
triglycerides, or liposomes. Aqueous injection suspensions may
contain substances which increase the viscosity of the suspension,
such as sodium carboxymethyl cellulose, sorbitol, or dextran.
Optionally, the suspension may also contain suitable stabilizers or
agents which increase the solubility of the compounds to allow for
the preparation of highly concentrated solutions.
[0208] Pharmaceutical preparations for oral use can be obtained by
combining the active compounds with solid excipient, optionally
grinding a resulting mixture, and processing the mixture of
granules, after adding suitable auxiliaries, if desired, to obtain
tablets or dragee cores. Suitable excipients are, in particular,
fillers such as sugars, including lactose, sucrose, mannitol, or
sorbitol; cellulose preparations such as, for example, maize
starch, wheat starch, rice starch, potato starch, gelatin, gum
tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium
carboxymethylcellulose, and/or polyvinyl-pyrrolidone (PVP). If
desired, disintegrating agents may be added, such as the
cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt
thereof such as sodium alginate. Such compositions may be prepared
by any of the methods of pharmacy but all methods include the step
of bringing into association one or more chemical agents as
described above with the carrier which constitutes one or more
necessary ingredients. In general, the pharmaceutical compositions
of the present invention may be manufactured in a manner that is
itself known, e.g. by means of conventional mixing, dissolving,
granulating, dragee-making, levigating, emulsifying, encapsulating,
entrapping or lyophilising processes.
[0209] Dragee cores are provided with suitable coatings. For this
purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer
solutions, and suitable organic solvents or solvent mixtures.
Dyestuffs or pigments may be added to the tablets or dragee
coatings for identification or to characterize different
combinations of active compound doses.
[0210] Pharmaceutical compositions which can be used orally include
push-fit capsules made of gelatin, as well as soft, sealed capsules
made of gelatin and a plasticizer, such as glycerol or sorbitol.
The push-fit capsules can contain the active ingredients in
admixture with filler such as lactose, binders such as starches,
and/or lubricants such as talc or magnesium stearate and,
optionally, stabilizers. In soft capsules, the active compounds may
be dissolved or suspended in suitable liquids, such as fatty oils,
liquid paraffin, or liquid polyethylene glycols. In addition,
stabilizers may be added.
[0211] Dosage forms of the chemical agents of the invention may
also include injecting or implanting controlled releasing devices
designed specifically for this purpose or other forms of implants
modified to act additionally in this fashion. Controlled release of
an agent of the invention may be effected by coating the same, for
example, with hydrophobic polymers including acrylic resins, waxes,
higher aliphatic alcohols, polylactic and polyglycolic acids and
certain cellulose derivatives such as hydroxypropyhnethyl
cellulose. In addition, controlled release may be effected by using
other polymer matrices, liposomes and/or microspheres.
[0212] Chemical agents of the invention may be provided as salts
with pharmaceutically compatible counterions. Pharmaceutically
compatible salts may be formed with many acids, including but not
limited to hydrochloric, sulphuric, acetic, lactic, tartaric,
malic, succinic, etc. Salts tend to be more soluble in aqueous or
other protonic solvents that are the corresponding free base
forms.
[0213] For any chemical agent used in the method of the invention,
the therapeutically effective dose can be estimated initially from
cell culture assays. For example, a dose can be formulated in
animal models to achieve a circulating concentration range that
includes the IC50 as determined in cell culture (e.g. the
concentration of a test agent, which achieves a half-maximal
inhibition of infection or colonization or presence of a biological
entity). Such information can be used to more accurately determine
useful doses in humans.
[0214] Toxicity and therapeutic efficacy of such chemical agents
can be determined by standard pharmaceutical procedures in cell
cultures or experimental animals, e.g. for determining the LD50
(the dose lethal to 50% of the population) and the ED50 (the dose
therapeutically effective in 50% of the population). The dose ratio
between toxic and therapeutic effects is the therapeutic index and
it can be expressed as the ratio LD50/ED50. Compounds that exhibit
large therapeutic indices are preferred. The data obtained from
these cell culture assays and animal studies can be used in
formulating a range of dosages for use in humans. The dosage of
such compounds lies preferably within a range of circulating
concentrations that include the ED50 with little or no toxicity.
The dosage may vary within this range depending upon the dosage
form employed and the route of administration utilized. The exact
formulation, route of administration and dosage can be chosen by
the individual physician in view of the patient's condition (see
for example Fingl et al., 1975, in "The Pharmacological Basis of
Therapeutics", Ch. 1 p1).
[0215] Dosage amount and interval may be adjusted individually to
provide plasma levels of the active agent which are sufficient to
maintain symptom-ameliorating effects. Usual patient dosages for
systemic administration range from 1-2000 mg/day, commonly from
1-250 mg/day, and typically from 10-150 mg/day. Stated in terms of
patient body weight, usual dosages range from 0.02-25 mg/kg/day,
commonly from 0.02-3 mg/kg/day, typically from 0.2-1.5 mg/kg/day.
Stated in terms of patient body surface areas, usual dosages range
from 0.5-1200 mg/m.sup.2/day, commonly from 0.5-150 mg/m.sup.2/day,
typically from 5-100 mg/m.sup.2/day.
[0216] Alternately, one may administer the compound in a local
rather than systemic manner, for example, via injection of the
compound directly into a tissue, often in a depot or sustained
release formulation. Furthermore, one may administer the drug in a
targeted drug delivery system, for example, in a liposome coated
with tissue-specific antibody. The liposomes will be targeted to
and taken up selectively by the tissue. In cases of local
administration or selective uptake, the effective local
concentration of the agent may not be related to plasma
concentration.
[0217] The chemical agents of the invention can also be delivered
topically. For topical administration, a composition containing
between 0.001-5% or more chemical agent is generally suitable.
Regions for topical administration include the skin surface and
also mucous membrane tissues of the vagina, rectum, nose, mouth,
and throat. Compositions for topical administration via the skin
and mucous membranes should not give rise to signs of irritation,
such as swelling or redness.
[0218] The topical composition may include a pharmaceutically
acceptable carrier adapted for topical administration. Thus, the
composition may take the form of a suspension, solution, ointment,
lotion, sexual lubricant, cream, foam, aerosol, spray, suppository,
implant, inhalant, tablet, capsule, dry powder, syrup, balm or
lozenge, for example. Methods for preparing such compositions are
well known in the pharmaceutical industry.
[0219] In one embodiment, the topical composition is administered
topically to a subject, e.g. by the direct laying on or spreading
of the composition on the epidermal or epithelial tissue of the
subject, or transdermally via a "patch". Such compositions include,
for example, lotions, creams, solutions, gels and solids. Suitable
carriers for topical administration preferably remain in place on
the skin as a continuous film, and resist being removed by
perspiration or immersion in water. Generally, the carrier is
organic in nature and capable of having dispersed or dissolved
therein a chemical agent of the invention. The carrier may include
pharmaceutically-acceptable emolients, emulsifiers, thickening
agents, solvents and the like.
[0220] The invention also features a process for separating
macrocyclic diterpenes from a biomass containing same, said process
comprising contacting the biomass with an aqueous solvent for a
time and under conditions sufficient to extract the macrocyclic
diterpenes into said solvent.
[0221] The aqueous solvent is preferably water.
[0222] Suitably, the biomass is derived from a plant, which is
preferably a member of the Euphorbiaceae family of plants or
botanical or horticultural relatives of such plants. Matter from
the plant (e.g. foliage, stems, roots, seeds, bark, etc.) is
preferably cut, macerated or mulched to increase the surface area
of the plant matter for aqueous extraction of the macrocyclic
diterpenes.
[0223] The process preferably further comprises adsorbing the
macrocyclic diterpenes to a non-ionic adsorbent, which is suitably
a non-ionic porous synthetic adsorbent. Among the non-ionic porous
synthetic adsorbents that can be used for the purposes of the
present invention include, but are not restricted to, aromatic
copolymers mainly composed of styrene and divinylbenzene, and
methacrylic copolymers mainly composed of monomethacrylate and
dimethacrylate. Such non-ionic porous synthetic adsorbents which
comprise, as the basic structure, aromatic copolymers mainly
composed of styrene and divinylbenzene include, for example, Diaion
HP10, HP20, HP21, HP30, HP40, HP50, SP850, and SP205 (trade names:
Mitsubishi Chemical Corp.), and Amberlite XAD-2, XAD4, (trade
names: Rohm and Haas Co.). Examples of non-ionic porous synthetic
adsorbent which comprise, as the basic structure, methacrylic
copolymer mainly composed of monomethacrylate and dimethacrylate
are Diaion HP2MG, Amberlite XAD-7, XAD-8 and XAD-16 and others.
[0224] Preferably, the process further comprises eluting
macrocyclic diterpenes from the non-ionic adsorbent with water and
water-soluble organic solvent(s).
[0225] The treatment may be conducted by a batch method using water
and water-soluble organic solvent(s) which dissolve macrocyclic
diterpenes, or may also be conducted continuously or in batch using
a column chromatography method.
[0226] Examples of a water-soluble organic solvent which may be
used in the present invention are alcohols such as methanol,
ethanol, n-propyl alcohol, isopropyl alcohol, and tert-butanol,
ethers such as dioxane and tetrahydrofuran, ketones such as
acetone, amides such as dimethylformamide, sulfur-containing
compounds such as dimethylsulfoxide. Two or more of such organic
solvents may be mixed for use. In addition, solvents less soluble
in water, for example, alcohols such as n-butanol, esters such as
methyl formate and methyl acetate, and ketones such as methyl ethyl
ketone may also be used to the extent that it does not separate
during development. Particularly preferred water-soluble organic
solvents are alcohols, in particular, methanol, ethanol, propyl
alcohol, and the like. Furthermore, different kinds of solvent may
also be used sequentially for development.
[0227] Macrocyclic diterpenes can be further purified using media
and techniques which separate compounds on the basis of molecular
size and/or polarity. In a preferred embodiment of this type, the
macrocyclic diterpenes are separated using Sephadex HL-20 resin and
preferably using water and water-soluble organic solvent(s) for
development.
[0228] The present invention contemplates the use of the chemical
agents of the invention for modulating the expression of genes
under the control of promoters whose activity is modulated by said
chemical agents. Thus, in one embodiment, the chemical agents of
the present invention find particular utility in modulating and
preferably in promoting or activating the expression of gene
therapy reagents (e.g. peptides, polypeptides, proteins, antisense
RNA, sense RNA) under the control of such modulatable
promoters.
[0229] The term "gene therapy" refers generally to the therapeutic
replacement, augmenting or silencing of an endogenous gene
activity, which is typically effected by gene transfer.
[0230] "Gene transfer" or "gene delivery" as used herein refer to
methods or systems for reliably inserting exogenous nucleic acids,
typically DNA, into host cells. Such methods can result in
transient expression of non-integrated transferred nucleic acids,
extrachromosomal replication and expression of transferred
replicons (e.g. episomes), or integration of transferred genetic
material into the genomic DNA of host cells. A number of systems
have been developed for gene transfer into mammalian cells. See,
e.g., U.S. Pat. No. 5,399,346.
[0231] As used herein, "exogenous" means any nucleic acid that is
introduced into an organism or cell.
[0232] Promoters suitable for the present invention include viral
and non-viral promoter, which are preferably inducible.
Non-limiting examples of inducible non-viral promoters include
metallothionein promoters, and metallothionein-related promoters.
Non-limiting examples of inducible viral promoters effective for
use in gene transfer vectors of the present invention include, but
are not limited to, mouse mammary tumour virus promoters, CMV
(cytomegalovirus), SV40 (simian virus 40), RSV (Rous sarcoma
virus), HSV (herpes simplex virus), EBV (Epstein-Barr virus),
retroviral promoters and adenoviral promoters.
[0233] By "vector" is meant any genetic element, such as a plasmid,
phage, transposon, cosmid, chromosome, virus, virion, etc., which
is capable of replication when associated with the proper control
elements and which can transfer gene sequences between cells. Thus,
the term includes cloning and expression vehicles, as well as viral
vectors.
[0234] The ability of certain viruses to infect cells or enter
cells via receptor-mediated endocytosis, and to integrate into host
cell genome and express viral genes stably and efficiently have
made them attractive candidates for the transfer of foreign genes
into mammalian cells. Preferred gene therapy vectors of the present
invention are viral vectors.
[0235] A viral-based vector comprises: (1) nucleic acid of, or
corresponding to at least a portion of, the genome of a virus,
which portion is capable of directing the expression of a DNA
sequence; and (2) a DNA sequence encoding an expression product
(e.g. polypeptide or transcript), operably linked to the viral
nucleic acid and capable of being expressed as a functional gene
product in the target cell. The recombinant viral vectors of the
present invention may be derived from a variety of viral nucleic
acids known to one skilled in the art, e.g. the genomes of
adenovirus, adeno-associated virus, HSV, alphavirus, vaccinia
virus, and other viruses, including RNA and DNA viruses.
[0236] Particularly preferred viral vectors are those that can
accept foreign genetic material and can infect an extended range of
cells. Adenoviruses do not integrate their genetic material into
the host genome and therefore do not require host replication for
gene expression, making them ideally suited for rapid, efficient,
heterologous gene expression. Techniques for preparing
replication-defective infective viruses are well known in the art.
Preferably a viral delivery system including the virion is free of
undesirable contaminants, such as defective interfering viral
particles or endotoxins and other pyrogens such that it will not
cause any inappropriate reactions in the cell, animal or individual
receiving the adenoviral vector construct. A preferred means of
purifying the vector involves the use of buoyant density gradients,
such as cesium chloride gradient centrifugation.
[0237] Adenovirus is a particularly preferred gene therapy vector
Particularly preferred Adenoviral vector of the present invention
include commercially available retroviral expression vectors
including but not limited to pLAPSN, pLEGFP-C1, pLEGFP-N1, pLHCX,
pLIB, pLNCX, pLNCX2, pLNHX, pLPCX, pLXIN, pLXRN, pLXSN, pMSCVhyg,
pMSCVneo, pMSCVpuro, pSIR, and pVSV-G.
[0238] The present invention further contemplates the use of
Adeno-associated virus (AAV) is vector system for use in the
present invention. Details concerning the generation and use of
recombinant AAV vectors are described in U.S. Pat. Nos. 5,139,941
and 4,797,368, each incorporated herein by reference. The present
invention further contemplates gene therapy using retroviral
vectors such as an engineered variant of the Moloney murine
leukemia virus.
[0239] In another embodiment of the present invention, the viral
vector may be derived from other viral vectors such as vaccinia
virus, sindbis virus, cytomegalovirus and herpes simplex virus.
[0240] The present invention further contemplates the use of gene
therapy vectors derived from an RNA virus such as but not limited
to the following: members of the family Reoviridae, including the
genus Orthoreovirus (multiple serotypes of both mammalian and avian
retroviruses), the genus Orbivirus (Bluetongue virus, Eugenangee
virus, Kemerovo virus, African horse sickness virus, and Colorado
Tick Fever virus), the genus Rotavirus (human rotavirus, Nebraska
calf diarrhea virus, murine rotavirus, simian rotavirus, bovine or
ovine rotavirus, avian rotavirus); the family Retrovirideae
including the genus Lentivirus (Human Immunodeficiency Virus), the
family Picomaviridae, including the genus Enterovirus (poliovirus,
Coxsackie virus A and B, enteric cytopathic human orphan (ECHO)
viruses, hepatitis A virus, Simian enteroviruses, Murine
encephalomyelitis (ME) viruses, Poliovirus muris, Bovine
enteroviruses, Porcine enteroviruses, the genus Cardiovirus
(Encephalomyocarditis virus (EMC), Mengovirus), the genus
Rhinovirus (Human rhinoviruses including at least 113 subtypes;
other rhinoviruses), the genus Apthovirus (Foot and Mouth disease
(FMDV); the family Calciviridae, including Vesicular exanthema of
swine virus, San Miguel sea lion virus, Feline picornavirus and
Norwalk virus; the family Togaviridae, including the genus
Alphavirus (Eastern equine encephalitis virus, Semliki forest
virus, Sindbis virus, Chikungunya virus, O'Nyong-Nyong virus, Ross
river virus, Venezuelan equine encephalitis virus, Western equine
encephalitis virus), the genus Flavirius (Mosquito borne yellow
fever virus, Dengue virus, Japanese encephalitis virus, St. Louis
encephalitis virus, Murray Valley encephalitis virus, West Nile
virus, Kunjin virus, Central European tick borne virus, Far Eastern
tick borne virus, Kyasanur forest virus, Louping III virus,
Powassan virus, Omsk hemorrhagic fever virus), the genus Rubivirus
(Rubella virus), the genus Pestivirus (Mucosal disease virus, Hog
cholera virus, Border disease virus); the family Bunyaviridae,
including the genus Bunyvirus (Bunyamwera and related viruses,
California encephalitis group viruses), the genus Phlebovirus
(Sandfly fever Sicilian virus, Rift Valley fever virus), the genus
Nairovirus (Crimean-Congo hemorrhagic fever virus, Nairobi sheep
disease virus), and the genus Uukuvirus (Uukuniemi and related
viruses); the family Orthomyxoviridae, including the genus
Influenza virus (Influenza virus type A, many human subtypes);
Swine influenza virus, and Avian and Equine Influenza viruses;
influenza type B (many human subtypes), and influenza type C
(possible separate genus); the family paramyxoviridae, including
the genus Paramyxovirus (Parainfluenza virus type 1, Sendai virus,
Hemadsorption virus, Parainfluenza viruses types 2 to 5, Newcastle
Disease Virus, Mumps virus), the genus Morbillivirus (Measles
virus, subacute sclerosing panencephalitis virus, distemper virus,
Rinderpest virus), the genus Pneumovirus (respiratory syncytial
virus (RSV), Bovine respiratory syncytial virus and Pneumonia virus
of mice); forest virus, Sindbis virus, Chikungunya virus,
O'Nyong-Nyong virus, Ross river virus, Venezuelan equine
encephalitis virus, Western equine encephalitis virus), the genus
Flavirius (Mosquito borne yellow fever virus, Dengue virus,
Japanese encephalitis virus, St. Louis encephalitis virus, Murray
Valley encephalitis virus, West Nile virus, Kunjin virus, Central
European tick borne virus, Far Eastern tick borne virus, Kyasanur
forest virus, Louping III virus, Powassan virus, Omsk hemorrhagic
fever virus), the genus Rubivirus (Rubella virus), the genus
Pestivirus (Mucosal disease virus, Hog cholera virus, Border
disease virus); the family Bunyaviridae, including the genus
Bunyvirus (Bunyamwera and related viruses, California encephalitis
group viruses), the genus Phlebovirus (Sandfly fever Sicilian
virus, Rift Valley fever virus), the genus Nairovirus
(Crimean-Congo hemorrhagic fever virus, Nairobi sheep disease
virus), and the genus Uukuvirus (Uukuniemi and related viruses);
the family Orthomyxoviridae, including the genus Influenza virus
(Influenza virus type A, many human subtypes); Swine influenza
virus, and Avian and Equine Influenza viruses; influenza type B
(many human subtypes), and influenza type C (possible separate
genus); the family paramyxoviridae, including the genus
Paramyxovirus (Parainfluenza virus type 1, Sendai virus,
Hemadsorption virus, Parainfluenza viruses types 2 to 5, Newcastle
Disease Virus, Mumps virus), the genus Morbillivirus (Measles
virus, subacute sclerosing panencephalitis virus, distemper virus,
Rinderpest virus), the genus Pneumovirus (respiratory syncytial
virus (RSV), Bovine respiratory syncytial virus and Pneumonia virus
of mice); the family Rhabdoviridae, including the genus
Vesiculovirus (VSV), Chandipura virus, Flanders-Hart Park virus),
the genus Lyssavirus (Rabies virus), fish Rhabdoviruses, and two
probable Rhabdoviruses (Marburg virus and Ebola virus); the family
Arenaviridae, including Lymphocytic choriomeningitis virus (LCM),
Tacaribe virus complex, and Lassa virus; the family Coronoaviridae,
including Infectious Bronchitis Virus (IBV), Mouse Hepatitis virus,
Human enteric corona virus, and Feline infectious peritonitis
(Feline coronavirus).
[0241] The present invention further envisions the use of viral
vectors derived from the following DNA viruses including but not
limited to the family Poxviridae, including the genus Orthopoxvirus
(Variola major, Variola minor, Monkey pox Vaccinia, Cowpox,
Buffalopox, Rabbitpox, Ectromelia), the genus Leporipoxvirus
(Myxoma, Fibroma), the genus Avipoxvirus (Fowlpox, other avian
poxvirus), the genus Capripoxvirus (sheeppox, goatpox), the genus
Suipoxvirus (Swinepox), the genus Parapoxvirus (contagious postular
dermatitis virus, pseudocowpox, bovine papular stomatitis virus);
the family Iridoviridae (African swine fever virus, Frog viruses 2
and 3, Lymphocystis virus of fish); the family Herpesviridae,
including the alpha-Herpesviruses (Herpes Simplex Types 1 and 2,
Varicella-Zoster, Equine abortion virus, Equine herpes virus 2 and
3, pseudorabies virus, infectious bovine keratoconjunctivitis
virus, infectious bovine rhinotracheitis virus, feline
rhinotracheitis virus, infectious laryngotracheitis virus) the
Beta-herpesviruses (Human cytomegalovirus and cytomegaloviruses of
swine, monkeys and rodents); the gamma-herpesviruses (Epstein-Barr
virus (EBV), Marek's disease virus, Herpes saimiri, Herpesvirus
ateles, Herpesvirus sylvilagus, guinea pig herpes virus, Lucke
tumor virus); the family Adenoviridae, including the genus
Mastadenovirus (Human subgroups A,B,C,D,E and ungrouped; simian
adenoviruses (at least 23 serotypes), infectious canine hepatitis,
and adenoviruses of cattle, pigs, sheep, frogs and many other
species, the genus Aviadenovirus (Avian adenoviruses); and
non-cultivatable adenoviruses; the family Papoviridae, including
the genus Papillomavirus (Human papilloma viruses, bovine papilloma
viruses, Shope rabbit papilloma virus, and various pathogenic
papilloma viruses of other species), the genus Polyomavirus
(polyomavirus, Simian vacuolating agent (SV-40), Rabbit vacuolating
agent (RKV), K virus, BK virus, JC virus, and other primate polyoma
viruses such as Lymphotrophic papilloma virus); the family
Parvoviridae including the genus Adeno-associated viruses, the
genus Parvovirus (Feline panleukopenia virus, bovine parvovirus,
canine parvovirus, Aleutian mink disease virus, etc.). Finally, DNA
viruses may include viruses which do not fit into the above
families such as Kuru and Creutzfeldt-Jacob disease viruses and
chronic infectious neuropathic agents (CHINA virus).
[0242] The present invention is further directed to the use of
defective hepatitis B viruses as gene therapy reagent. In a related
aspect of the present invention, the gene therapy vector can be
HSV. In various embodiments of the invention, DNA is delivered to
an animal as an expression construct. In order to effect expression
of a gene construct, the expression construct must be delivered
into a cell. As described herein, the preferred mechanism for DNA
delivery is via viral infection, where the expression construct is
encapsidated in an infectious viral particle. However, several
non-viral methods for the transfer of expression constructs into
cells also are contemplated by the present invention. In one
embodiment of the present invention, the expression construct may
consist only of naked recombinant DNA or plasmids. Transfection of
the construct may be performed by any of the methods mentioned
which physically or chemically permeabilize the cell membrane. One
or other of these techniques may be successfully adapted for in
vivo or ex vivo use, In a further embodiment of the invention, the
expression construct may be transfected as a liposome. Also it is
further contemplated that an expression construct complexed with
Lipofectamine (Gibco BRL) may be used to facilitate non-viral
transfection.
[0243] The introduction of a vector into a cell of interest may be
effected by methods known to one skilled in the art, such as
electroporation, DEAE Dextran, cationic liposome fusion, protoplast
fusion, DNA-coated microprojectile bombardment, injection with
recombinant replication-defective viruses, homologous
recombination, and naked DNA transfer by, for example, intravesical
instillation. It will be appreciated by those skilled in the art
that any of these methods of gene transfer may be combined.
[0244] The present invention is further described by the following
non-limiting Examples.
EXAMPLE 1
PKC Activation: Kinase Activity of PKC as Measured by Enzyme
Assay
[0245] Preparation of Chemical Fractions from E. peplus
[0246] Sap from E. peplus plants was collected, stored at
-20.degree. C., thawed and stored at 4.degree. C. for 1 week prior
to use. The H fraction was prepared from frozen sap by thin layer
chromatography (TLC) as described in International Patent
Application No. PCT/AU98/00656 and was stored as dried
silica-associated material at 4.degree. C. This material was
enriched in jatrophanes and pepluanes. One to two months prior to
use, the material was dissolved in ethylene glycol dimethyl ether
(DME) and stored at 4.degree. C. The concentrations were determined
from the dry weight of the material. For PKC assays, crude sap
(PEP001) and the PEP004 fraction was ether extracted twice to
produce an ether-soluble fraction enriched in diterpenes, namely,
ingenanes, jatrophanes and pepluanes. The remaining water soluble
fraction was also used. An ingenane fraction was prepared from the
ether-soluble extract by TLC as described in International Patent
Application No. PCT/AU98/00656.
[0247] PKC Assay
[0248] The conventional and novel protein kinase C (PKC) isoforms,
in their unstimulated state, are inactive as kinases. The C1 domain
of these PKCs contains an autoinhibitory, pseudosubstrate site that
binds to the substrate site (C4 domain) and inactivates the kinase
functionality of the protein. Activation of PKC results from
binding of diacylglycerol (DAG) to the C1 domain, which, via
multiple phosphorylation events and conformational changes to the
protein, ultimately leads to the release of PKC autoinhibition. TPA
and other related compounds have been shown to bind to the C1
domain of various PKC isoforms and presumably by similar means as
DAG, lead to their activation.
[0249] The kinase activity of rat brain PKC (Promega) was
determined using the Peptag.TM. Non-Radioactive Protein Kinase Kit
(Promega). Using agarose gel electrophoresis the technique
visualises the opposing electrostatic charge of a fluorescently
labeled peptide (PLSRTLSVAAK) compared to the phosphorylated
version of the same peptide.
[0250] The results of an assay of PKC with the fluorescent
substrate (PepTag) are shown in FIG. 1. The reaction mixture was
separated by gel electrophoresis, showing migration of the
unreacted substrate (a) to the anode (top), and the product (b),
which is more negatively charged because of phosphorylation by PKC,
moving towards the cathode (bottom). The positive control activator
(phosphatidyl serine) supplied by the manufacturer (lane 2) showed
strong activation compared with PKC and substrate alone (lane 1).
Various dilutions of TPA also showed activation of PKC (lanes 3, 4
and 5).
[0251] An ether extract of E. peplus sap, reconstituted in
dimethoxyethane (DME) and incubated with PKC at a final dilution of
1 in 5 relative to the sap, gave a significant level of action
(lane 7), as did the crude sap itself (lane 9). In the latter case,
however, both the substrate and product (band c, lane 9) were found
further towards the cathode. This result was interpreted as being
due to a carboxypeptidase activity in the crude sap, cleaving the
C-terminal, positively-charged lysine from the substrate peptide.
This was confirmed by the finding that the aqueous layer from ether
extraction had minimal PKC-activating ability, but altered
migration of the substrate in the same way as the crude sap (lane
8). DME itself had no activity (lane 10).
[0252] FIG. 2 shows the results of testing fractionated materials
simultaneously with negative (lane 1) and positive controls (lane
2). Fraction H (mixture of jatrophanes and pepluanes) showed a low
activity (lane 3), seen as a halo of product (arrow) moving away
from the unreacted substrate. A similar result was found for the
ingenane fraction (lane 4).
[0253] All of the E. peplus fractions are tested for activation of
all the available protein kinase enzymes using the peptide-based
fluorescent tag test described above. The isoenzymes available for
this experiment (Panevera) were .alpha., .beta.1 , .beta.11,
.gamma., .delta., .epsilon., .eta. and .zeta..
[0254] Essentially, the kinase activity of the PKC sample was
assessed before stimulation (Negative Control) and after
stimulation with PEP001, phosphatidyl serine (an acid-lipid, known
to activate PKC, provided by Promega; Positive Control) and TPA (20
.mu.g/mL). The results presented in FIG. 3 indicate that PEP001, at
dilutions of 1:125 and 1:500, activates PKC to a similar level as
phosphatidyl serine (200 .mu.g/mL) and to a greater level than TPA
(20 .mu.g/mL). From this experiment, it is clear that the PEP001
activates PKC.
EXAMPLE 2
PKC Activation: Translocation of PKC
[0255] Activation of PKC can also be demonstrated by a simple
fluorescence microscopy-based assay. Upon activation, PKC is known
to translocate from the cytoplasm to the plasma membrane of the
cell. By fusing PKC enzymes to the green fluorescent protein (GFP)
or enhanced GFP (EGFP), activation of the PKC can be detected by
the movement of diffuse cytoplasmic GFP to a ring of fluorescence
associated with the plasma membrane. Using this assay, crude E.
peplus extract has been shown to activate PKC.beta. and
PKC.gamma..
[0256] MM96L cells were first transfected using a
commercially-available kit (Qiagen Effectine Transfection Kit) with
a PKC-GFP expression vector (Clontech;
http://www.clontech.com/gfp/) and allowed to produce the PKC-GFP
protein for 24 hr. The cells were then treated with crude E. peplus
extract and TPA and observed under a fluorescent microscope (488 nm
excitation). Two controls were used--no DNA, which allows for the
identification of non-transfected cells, and no drugs, which allows
for the calculation of transfection efficiency and the
identification of transfected cells without PKC activation.
pPKC.beta.-EGFP and pPKC.gamma.-EGFP were tested, and crude E.
peplus extract was shown to induce movement of the fluorescence
from the cytosol to the plasma membrane, indicating that crude E.
peplus extract activated these PKC enzymes. The results are
illustrated in FIGS. 4A and 4B, which respectively show expression
of PKCP in the absence of any drug and after exposure to crude E.
peplus extract for 2 hr.
[0257] In another experiment, translocation of individual PKC
isoforrns was observed using fluorescence microscopy and used as an
indication of activation by PEP003 and PEP005.
[0258] Five EGFP-PKC isoforms (Clontech) were available for this
experiment, enabling the screening of the three predominant PKC
families (i.e. classical, novel and atypical PKCs). The members of
the various PKC families are .alpha., .beta., and .gamma.
(classical), .theta. (novel) and .zeta. (atypical).
[0259] HeLa cells were plated out in a 24-well plate containing
coverslips and transfected with PKC isoforms fused to EGFP, using a
commercially available effectine-transfection kit (QIAGEN, Pty.
Ltd.). Cells were exposed to the transfection reagents for 16-24
hr. Subsequently, transfected cells were treated for one hour with
TPA (100 ng/mL), bryostatin-1 (5 pg/mL), PEP003 (2.25 .mu.g/mL; 5
.mu.M) or PEP005 (670 .mu.g/mL) 1.5 .mu.M). Following treatment,
cells were fixed on coverslips and mounted on glass slides. The
slides were subsequently examined visually by fluorescence
microscopy, photographed, and over 150 cells were
counted/treatment/PKC isoform. Counted cells were classified
according to the localisation of the PKC-EGFP fluorescence as
either cytoplasmic or plasma membrane using ImagePro.TM. 4.1 (FIG.
5). Several cells also showed localisation to the Golgi, or
similarly located cellular structure (FIG. 5). The number of these
cells was also counted. Results are presented as an average and
standard deviation of percentages of cells (Table 1).
[0260] The results presented in FIG. 6 show that PKC .alpha.,
.beta. and .gamma. are translocated from the cytoplasm to the
plasma membrane in response to treatment with PEP003, PEP005 and
TPA but not with bryostatin-1. As expected, the
diacylglycerol-independent PKC.zeta. is not translocated in
response to any treatment. PKC.theta. is translocated in response
to PEP003, TPA and bryostatin-1, however, PEP005 does not induce
any change in the isoenzymes localization. The results also show
that treatment of PKC.alpha. and .gamma. transfected cells with
TPA, PEP003 and PEP005 leads to an increase in the number of cells
displaying Golgi-like fluorescence. PKC.beta. transfected HeLa
cells treated with TPA also show an increase in Golgi-Tike
fluorescence. In contrast, treatment with PEP005 and bryostatin-1
decreases the number of cells with PKC.beta. concentrated in the
Golgi. The number of PKC.theta. transfected HeLa cells with
Golgi-like localization is increased in response to all
treatments.
[0261] The above results indicate that PEP003 and PEP005 induce
translocation of the classical and novel PKC isoforms tested,
suggesting that these compounds activate members of the classical
and novel PKC families. TPA, Bryostatin-1, PEP003 and PEP005 fail
to induce translocation of PKC.zeta., suggesting that PEP003 and
PEP005 do not activate members of the atypical PKC family.
Furthermore, TPA, Bryostatin-1, PEP003 and PEP005 display
differences in their ability to induce translocation of the
specific PKC isoforms to the plasma membrane and/or Golgi. These
differences may play a role in determining the different biological
actions of these compounds.
EXAMPLE 3
Binding of Compounds to PKC
[0262] A competition assay was performed to determine whether the
diterpene esters of the instant invention bind to the phorbol ester
binding site of PKC. This competition assay showed that 23 .mu.g/mL
PEP003 displaced >90% [3H]-phorbol dibutyrate from binding to
rat brain homogenate, used as a source of PKC (Gonzalez et al.,
1999). This binding was not blocked by co-incubation with
bisindolylmaleimide. These results show that PEP003 binds to the
phorbol ester binding site of PKC, and bisindolylmaleimide does
not.
EXAMPLE 4
Activation of Latent HIV Infection
[0263] The use of highly active anti-retroviral therapy such as
combinations of reverse transcriptase inhibitors and protease
inhibitors (HAART) has significantly prolonged the life of
individuals infected with HIV. However, the regimen is very
burdensome, requiring strict adherence to prevent a recurrence of
viraemia. Long-lived cells capable of actively transcribing virus,
such as CD4.sup.+ cells, act as a major latent reservoir and enable
the virus to avoid anti-retroviral chemotherapy or immune system
surveillance. There is, therefore, an urgent need to find an agent
which activates latent virus from the infected cells. Activated
virus could then be killed by aggressive anti-retroviral
chemotherapy and it has been hypothesized that immune system
surveillance could also be improved under these conditions. Such an
agent could have utility in other disease states in which virus is
sequestered in infected cells, e.g. herpes infections. Anti-cancer
agents have been widely investigated as potential anti-HIV agents.
Several PKC activators have been shown to activate latent
retroviruses. For example, PMA has been shown to activate latent
HIV in monocytes (Tobiume et al., 1998). However, PMA is a known
tumor promoter. A latently HIV-1 infected cell line (U1), derived
from the promonocytic cell line U937 after infection with HIV-1 LAI
strain, was used in these experiments. In the absence of
activation, no or little virus (measured as p24 production) is
produced by the U1 cell line. Phorbol esters are known to activate
virus production from these cells (Tobiume et al., 1998) and so
TPA/PMA was used as a positive control in these experiments.
[0264] U1 cells were cultured in RPMI-1640 medium supplemented with
10% fetal bovine serum, 10.sup.5 cells/mL were cultured for 20 hr
in the presence and absence of various concentrations of either the
phorbol ester TPA or crude E. peplus sap (PEP001) or PEP004 (HI)
derived therefrom. Supernatants were collected and viral
replication monitored by determination of the amounts of HIV p24
gag protein in the culture supernatants by ELISA, using a NEN Life
Science HIV-1 p24 ELISA kit. p24 values were calculated from OD
values using a standard curve.
[0265] TPA, the crude sap (PEP001) from E. peplus and the PEP004
fraction all activated HIV from U1 cells, as illustrated in FIG. 7.
The crude sap (PEP001) was 50 times less active than TPA. The
PEP004 fraction was 1000 times less active than TPA.
EXAMPLE 5
Lytic HIV Activity Inhibited by PEP003 and PEP004
[0266] The human immunodeficiency virus (HIV), a retrovirus, is the
cause of the fourth greatest killing disease in the world,
infecting more than 36 million people. A number of anti-retroviral
compounds have been approved for clinical use, but many HIV strains
have developed resistance to these drugs. There is clear and
immediate need for new anti-retroviral compounds.
[0267] Experiments were conducted to assess the effect of the
compounds of the instant invention on HIV-1 replication in acutely
infected T cells. Peripheral blood mononuclear cells (PBMC) were
obtained from non-HIV-1, non-HIV-2, non-Hepatitis B/C infected
donors, stimulated with phytohemagglutinin-M and grown in culture
media supplemented with 10 U/mL interleukin-2. The activated PBMC
were infected with 10 g (Low Titre) and 100 ng (High Titre) of
CA-p24 equivalents of the HIV-strain pNL4-3. Cells were infected
for two hr after which, the virus was removed and the cells were
washed with culture media. Equivalent numbers of cells were seeded
into 24 well plates and compounds were added to the cultured cells
that included: TPA at 8 riM and 80 nM, Ingenol at 280 nM, PEP003 at
500 nM, 50 nM and 5 nM, or PEP004 at dilutions of 1.times.10.sup.4
and 1.times.10.sup.5 from the stock (final concentrations). In
addition, uninfected activated PBMC were grown in the presence of
TPA (80 nM), Ingenol (280 nM), PEP003 (500 nM) and PEP004
(1.times.10.sup.4 dilution). Other cultures were neither infected
nor treated with any compound, or infected but not treated with any
compound. Supernatant was removed from each culture at day 0, 3, 7,
and 10. The amount of HIV-1 CA-p24 was determined using a
commercially available ELISA assay. Three independent experiments
were performed.
[0268] The data presented in FIGS. 8A-8D show that PEP003 reduced
virus replication kinetics in a dose-dependent manner. PEP003 at
concentrations of 500 nM, 50 nM and 5 nM reduced the replication
rate by approximately 99.9%, 95% and 47%, respectively, relative to
the untreated, infected cells. PEP004 at dilutions of
1.times.10.sup.4 and 1.times.10.sup.5 reduced the replication rate
by approximately 66% and 15%, respectively. Viral load seemed to
alter these results slightly, as higher initial inoculums of virus
reduced the total inhibition of PEP003 at 500 nM or 50 nM to
approximately 97% (t-test; p<0.001) or 88% (t-test; p<0.074),
respectively. The control compounds Ingenol (2.8 .mu.M) and TPA (80
nM or 8 nM) reduced HIV-1 replication rates by approximately 35%,
98% and 38%, respectively.
EXAMPLE 6
Enhancement of the Cytomegalovirus Promoter Activity as a Method
for Improving Gene Therapy
[0269] Viruses and viral promoters especially adenovirus and CMV
are used to deliver gene therapy in a range of human disease
conditions. Gene expression and, hence, therapeutic effect will be
enhanced if the promoters driving their transcription can be
activated further by an agent.
[0270] Human melanoma cells were infected with ten-fold dilutions
of adenovirus 5 in culture, treated with dilutions of PEP005,
PEP006, PEP008 and PEP010 and adenovirus replication determined 2
days later by immunhistochemical detection of virus-replicating
cells. Virus replication (enumerated as the number of stained cells
following successive incubations with adenovirus antibody,
peroxidase-conjugated protein A and peroxidase substrate) was
increased by 344% with 67 ng/mL PEP005, 256% with 295 ng/mL PEP006,
248% with 226 ng/mL PEP008 and 147% with 67.5 ng/mL PEP010.
[0271] The CMV promoter is commonly used to activate the
transcription of genes in constructs transfected into a variety of
cells, due to its strong transcriptional activity in a variety of
human cell types. The ability of TPA to increase this activity has
been demonstrated in cells undergoing non-productive infection with
an adenovirus construct (Christenson et al., 1999), thus raising
the possibility of increasing the production of a therapeutic
protein encoded by a similar construct.
[0272] Human melanoma cells (MM96L; 50,000 per microtiter well)
were treated with TPA or dilutions of crude E. peplus sap, infected
with a 1/20 dilution of a pool of adenovirus-5 expressing
.beta.-galactosidase driven by the CMV promoter. After incubation
for 20 hr, the wells were washed with 3.times. with PBS, 50 .mu.L
of chlorophenol red galactoside (GPRG) substrate solution added and
the absorbance at 540 nm read after 90 min. The inventors found TPA
(100 ng/mL) and crude E. peplus sap (diluted 1 in 10,000) both
induced the CMV promoter activity by >3-fold.
EXAMPLE 7
Activation of Innate Immune Responses: Induction of Neutrophil
Invasion in Skin
[0273] Neutrophils represent about 70% of peripheral white blood
cells in humans and play a pivotal role in inflammation and the
innate defense against disease (Mollinedo, 1999). Upon activation,
neutrophils release superoxide radicals and granules containing a
variety of enzymes and other compounds. These secretions are able
to destroy invading pathogens, but also result in inflammation and
associated tissue damage.
[0274] The inventors found that E. peplus sap causes accumulation
of neutrophils at the site of application, showing that E. peplus
sap is capable of recruiting neutrophils. A mixture of active
diterpenes obtained as an ether extract from E. peplus sap was
applied (2 .mu.L of 100 mg/mL in ethanol) on the skin of a nu/nu
mouse. After 24 hr, the animal was sacrificed and the skin fixed in
10% formalin for sectioning and hematoxylin/eosin staining. As
shown in FIGS. 9A and 9B, control skin showed normal skin structure
with few infiltrating monocytes. The treated skin showed large
numbers of infiltrating neutrophils, characterized by their
polymorphic nuclei. There was no evidence of gross damage to the
skin.
EXAMPLE 8
Neutrophil Infiltration Activity
[0275] Basal cell carcinoma (BCC) is the most common cancer in the
Caucasian population, with the highest annual incidence globally
having been recorded in Australia (Miller et al., 1994, Marks et
al., 1993). New developments have begun looking at treating
non-melanoma skin cancer (NMSC) using topical therapies. The
essence of this therapy may rely upon the induction of an
inflammatory response with infiltration of leucocytes, in
particular neutrophils.
[0276] To assess whether the compounds of the invention induce
neutrophil infiltration, an experiment was designed on C57BL/6J
mice. Twenty-four mice were divided into six groups of four mice
per group. In three of these groups the mice had a B16 melanoma
injected s.c. (2 sites per mouse, 5.times.10.sup.5 cells/site),
that was left to grow for 8 days to approximate tumor sizes of 5-8
mm in diameter. A single application of one of all three compounds
was then applied to the tumor or to normal skin. Each compound was
applied on two groups of mice, one with tumor and 1 without tumor.
The three compounds were PEP010 (2 .mu.L; 150 mM) in 10 .mu.L of
isopropanol gel (isopropyl alcohol 25% (w/w), propyl alcohol 25%
(w/w)) (vehicle), PEP009 (2 .mu.L of stock) in 10 .mu.L of vehicle
or vehicle alone as a control. One mouse from each group was then
sacrificed at either 4 hr, 24 hr, 48 hr or 144 hr post single
application of compound and then tissue excised and sections
prepared for histology.
[0277] The results at 4 hr show only minimal response with 1+
patchy neutrophils for both PEP010 on B16 tumor and PEP009 on
normal skin and 2+ neutrophils present for PEP009 on B16 tumor
(Table 2). At 24 hr, there are no neutrophils present in the
control groups with vehicle alone but a 4+ neutrophil infiltration
with PEP010 and PEP009 application, on both tumor and normal skin
(FIGS. 10A and 10B). In addition, 60-85% of the superficial tumor
cells were apoptotic or necrotic in the B16 groups. At 48 hr, there
was a similar pattern with a 4+ neutrophil presence with PEP010 and
PEP009 application while the control groups showed an absence of
neutrophils (FIGS. 10A and 10B). Along with the tumor cell necrosis
and apoptosis, there is also evidence of some neutrophil breakdown
at the 48 hour interval. The 144 hour group showed a lack of
neutrophils in the control group and a presence of 2-4+
neutrophils, which were mostly now degenerate in the PEP010 and
PEP009 groups. There was extensive necrosis of tumor and skin, and
clear signs of granulation tissue and early repair.
[0278] This study shows that the PEP010 and PEP009 induce a marked
inflammatory infiltrate of neutrophils as compared to vehicle alone
and this influx of polymorphonuclear cells may be significant in
altering the growth of certain skin cancers.
EXAMPLE 9
Activation of Innate Immune Responses: Induction of a Respiratory
Burst in Peripheral Blood Mononuclear Cells
[0279] Monocytes/macrophages are blood-borne and tissue cells which
are usually activated by T lymphocytes and antibodies. Upon
activation, they are able to phagocytose pathogens, release
superoxide radicals and are an important source of cytokines. Crude
E. peplus extract was shown to be able to induce the release of
superoxide radicals by use of a fluorescence-activated cell sorting
(FACS)-based method, in which superoxide radicals are detected by
the dye dihydroethidium. In addition, phagocytic activity was
activated by E. peplus, as shown by increased uptake of nitroblue
tetrazolium and adherence to plastic was increased by E. peplus;
this is believed to indicate activation and differentiation of
macrophages.
[0280] Human peripheral blood mononuclear cells (PBMC) prepared by
standard Ficoll separation comprise approximately 5% monocytes.
PBMC were incubated with dihydroethidium, a reduced form of the dye
which becomes fluorescent when oxidized by a respiratory burst,
then treated in 10% FCS-RPMI 1640 at 37.degree. C. for 15 min with
crude E. peplus extract diluted 1/1000 or 100 ng/mL TPA and
analyzed by flow cytometry using conventional methods (Handbook of
Flow Cytometry Methods, p. 151) The mean channel numbers for
fluorescence were 618 (controls) and 818 (E. peplus extract diluted
1/1000). These results, illustrated in FIGS. 11A and 11B, show that
the E. peplus extract induced intracellular oxidation of the dye,
typical of a respiratory burst. Phagocytic activity was determined
by a conventional method (Hudson and Hay, Practical Immunology,
3.sup.rd edition, p. 74). Cells were treated in 10% FCS-RPMI 1640
at 37.degree. C. for 20 min with introblue tetrazolium (NBT) and
crude E. peplus extract (PEP001) diluted 1/1000 or 100 ng/mL TPA,
followd by counting the number of blue-stained cells in a
haemocytometer. The average of three fields gave figures of <2%
(controls), 10% (TPA) and 8.7% (E. peplus sap) cells stained blue.
This demonstrates induction of phagocytic activity, part of the
normal response to infectious agents, by E. peplus sap, as shown by
uptake by cells of the blue NBT precipitate.
[0281] Experiments were also carried out using
2',7'-dichlorofluorescein diacetate (DCFH-DA) to measure the
production of H.sub.2O.sub.2.(J P Robinson, Oxidative burst
methods, in Handbook of Flow Cytometry Methods, Wiley-Liss Inc,
pp147-149, 1993). H.sub.2O.sub.2 oxidizes the non-fluorescent probe
(DCFH-DA) to a fluorescent probe that can then be detected by a
flow cytometer. Peripheral blood mononuclear cells (PBMC) were
extracted from a donor blood sample by lysis of heparinized blood
and used in a suspension of 1.times.10.sup.6/mL of phosphate
buffer, pH 7.3. The cells were then incubated with DCFH-DA (1
.mu.L/mL of 20 mM stock) for 15 minutes to allow it to be taken up
and trapped by hydrolysis with cellular esterases. The cells were
then stimulated by test compounds for 15 min at 37.degree. C.
Controls included in the experiment were unloaded control (cells
with no DCFH-DA) and loaded control (cells with DCFH-DA, but no
stimulation). These were used to monitor the non-specific oxidation
of unstimulated cells. The cells were then analyzed on the flow
cytometer (excitation at 488 nm, emission at 525.+-.20 nm), gating
each sample for individual cell populations-granulocytes, monocytes
and lymphocytes (Table 3).
[0282] All compounds except Bryostatin induced a respiratory burst,
the effect being strongest in granulocytes and monocytes compared
with lymphocytes. Similar results were obtained by measuring the
reduction, under the same conditions, of nitroblue tetrazolium,
measured as the proportion of purple-stained cells counted under
the microscope.
[0283] Evidence for the requirement of PKC activation was obtained
by addition of bisindolylnaleimide (10 .mu.g/mL or 1 .mu.g/mL) at
the same time as PEP005, PEP006, PEP008 and PEP010. This PKC
inhibitor blocked the respiratory burst seen with TPA and
PEP003.
[0284] Phagocytosis with Fluorescent Beads
[0285] Phagocytosis by peripheral blood mononuclear cells (PBMCs)
was assayed (Steinkamp et al., 1982) using 1 .mu.m Fluoresbrite.TM.
yellow-green fluorescent latex spheres (Polysciences, Inc.,
Warrington, Pa.). A sample of whole, heparinized blood was treated
with drug and 5.times.10E7 fluorescent latex beads in 10 .mu.L of
PBS added per mL of suspension. Cells were incubated and maintained
in suspension for 30 min by means of a shaker platform at
37.degree. C. The stirnulated and non-stimulated samples were then
lysed to isolate PBMCs. The PBMCs were run on the flow cytometer
measuring FITC (excitation at 488 nm, emission at 525.+-.20 nm),
gated for fluorescence (phagocytosed spheres) and light scatter
(cell size).
[0286] The data presented in Table 4 indicate that TPA, PEP006,
PEP008, PEP003 and PEP005 all stimulate phagocytosis in PBMCs.
EXAMPLE 10
Activation of Innate Antiviral Activity
[0287] Many viruses, including alphaviruses, are sensitive to
innate antiviral activities, which are often mediated by the
activation of interferon .alpha./.beta. responses (Antalis et al.,
1998). Such antiviral activities inhibit the ability of cells to
support viral replication. For many viral infections, including
those caused by Ross River virus, viral replication results in
virus-induced cytophathic effect (CPE) or cell death. Treatment of
human fibroblast cells with E. peplus ingenanes was shown to
activate antiviral activity and prevented CPE induced by an
alphavirus infection.
[0288] Human skin fibroblasts (10e4/well) were seeded in 96 well
plate and left overnight to adhere. An extract of E. peplus
ingenanes was added at 5 .mu.g/mL for 48 hr. An alphavirus (Ross
River virus, T48) was then added at a dose of 1, 10 and 100 cell
culture ID50 for 6 days (La Linn et al., 1996). The cytopathic
effect of the viral infection was assayed using crystal violet
staining. Protected cells stain violet, whereas cells which have
suffered CPE detach from the plate, leaving the well unstained.
Alphavirus-induced CPE was observed in treated cells only at a
100-fold greater dose of virus than was required to induce CPE in
untreated cells, indicating that a significant degree of protection
was conferred by the E. peplus extract.
EXAMPLE 11
Protection Against Intra-Peritoneal Streptococcal Infection: Effect
of PEP003 and PEP004 on Systemic Group A Streptococcal Infection in
Mice
[0289] Infection of humans with group A streptococcus
(Streptococcus pyogenes) (GAS) can cause a variety of clinical
manifestations including the relatively minor pharyngitis ("trep
throat" and impetigo (superficial skin infection) to more severe
invasive infections such as toxic shock syndrome and necrotizing
fasciitis, both of which, may lead to multisystem organ failure.
Lastly, the GAS post-infectious sequelae of rheumatic fever (RF),
rheumatic heart disease (RHD) and acute glomerulonephritis (AGN)
are a major problem in developing countries and indigenous
populations, particularly in Australian Aboriginals. Current
treatment for controlling GAS infection is with antibiotic therapy,
however, since continual high dose administration of antibiotic is
required in cases of repeated episodes of acute RF and the
development of RHD, poor compliance is often associated with the
persistence of these GAS-associated diseases. The development of a
vaccine against GAS infection would prevent GAS-associated diseases
including RF and RHD. In the absence of a vaccine, however, the
development of new drugs with improved anti-bacterial activity may
provide promising therapeutic agents.
[0290] The inventors' aim was to test the ability of the PEP003 and
PEP004 to systemically protect against GAS infection, in vivo. Mice
(n=10) were treated with 50 .mu.L of PEP003 (500 nM), PEP004 (1:100
dilution from stock) or control (PBS/10% acetone), 24 hr prior to
and thereafter i.p. challenge with live GAS. Two different strains
of mice (Quackenbush and B10.BR) and four different GAS strains
(NS-1, PL-1, 88/30 and M1) were used. Mice were monitored for two
weeks post-challenge and the percentage survival of mice
determined. Percentage survival in Quackenbush mice challenged with
PL-1 GAS was 70% (PEP003), 60% (PEP004) and 40% (control) (Table
5). Control mice that had been given the same successive treatment
of PEP003 and PEP004 (but not challenged) to rule out any potential
adverse side effects of the compounds were then also challenged
with PL-1; survival was 40%, 80%, and 20% for PEP003, PEP004 and
controls, respectively (Table 6). In the latter experiment, the
protective effect of PEP004 approached significance (p=0.06),
however, small numbers of mice were used (n=5). In Quackenbush mice
challenged with NS-1 GAS, survival was 50% for PEP003 and controls,
and 80% for PEP004 (Table 5). In B10.BR mice challenged with M1
GAS, survival was 10% for controls, 30% for PEP003 and 0% for
PEP004 (Table 5). In B10.BR mice challenged with 88/30 GAS,
survival was 20% for controls, 30% for PEP004 and 0% for PEP003
(Table 5). The data indicate a possible protective effect of PEP004
against systemic GAS challenge in Quackenbush mice. In addition,
these data indicate that a weekly treatment regimen of PEP003 and
PEP004 prior to GAS challenge may be more effective.
EXAMPLE 12
Anti-Escherichia coli Activity of PEP003: Activation of
Leucocytes
[0291] Blood was collected into a Sodium Heparin tube (Becton
Dickinson VACUTAINER) and leucocytes prepared by lysis of red blood
cells (Handbook of Flow Cytometry Methods. Robinson J P. Wiley-Liss
Inc 1993. Oxidative Burst Methods H.sub.2O.sub.2 DCF Assay by Flow
cytometry p 147-149). Leucocytes were resuspended and divided
equally into two tubes such that each tube contained
7.times.10.sup.6 peripheral blood cells (PBCs). Both tubes were
then centrifuged (Beckman, GS-6) at 1000 rpm for 10 minutes. The
supernatant was removed and the volume was then adjusted to 1 mL
with RPMI 1640 (Gibco BRL, antibiotic free supplemented with 10%
v/v fetal bovine serum. 100 .mu.L of PEP003 (to give a final
concentration of 23 .mu.g/mL containing 10% acetone was then added
to one tube and to the other, 100 .mu.L of PBS/10% Acetone. To each
tube, 10 .mu.L E. coli (competent cells, XL10-Blue, Stratagene) was
also added (to give a .about.1/100 dilution of a static culture).
Both tubes were vortexed then centrifuged (Beckman, GS-6) at 2500
rpm for 10 minutes. Lids were loosened and the tubes were incubated
at 37.degree. C./5% CO.sub.2.
[0292] Following 16 hr incubation, the tubes were vortexed. To
estimate the number of E. coli, 50 .mu.L was taken from both tubes
as well as the static starter culture (stored at 4.degree. C.),
transferred to Eppendorf tubes and centrifuged (Beckman, GS-15R) at
10,000 rpm for 10 minutes. Supernatant (.about.45 .mu.L) was
removed and the pellet resuspended in the remaining .about.5 .mu.L.
A smear was made on a glass slide using the 5 .mu.L bacterial
suspension and stained using Quick Dip (Histo.Labs, Riverstone,
Australia), a modified method of the Wright-Giemsa stain, which
stains bacteria blue. E. coli were counted using a conventional
light microscope (.times. 400) with an eyepiece micrometer (100
.mu.m.times.100 .mu.m). This count was then adjusted to give a
total count in the smear (area=12.5.times.10.sup.5 .mu.m.sup.2) and
expressed as the number of E. coli per mL. Another method of
measuring growth of E. coli was to read the absorbance (595 nm) of
the supernatant.
[0293] The results presented in FIGS. 12 and 13 show that treatment
of leucocytes with PEP003 results in a significant reduction in
bacterial numbers.
EXAMPLE 13
Treatment of Ringworm
[0294] Ringworm is a subcutaneous mycosis or dermatophytosis caused
by fungi of the species Trichophyton, Microsporum and
Epidermophyton, in which the infection is confined to the
keratinous structures of the body. A two week old ringworm lesion,
determined to be Trichophyton mentagrophytes var. mentagrophytes by
culture, on the volar surface of the forearm of an adult male human
was treated with a single topical application of crude E. peplus
extract and was shown to resolve after seven days. Resolution of
such lesions in the absence of treatment does occur, but is
considered extremely rare.
EXAMPLE 14
Treatment for Bites of Blood-Sucking Insects
[0295] The bites of blood sucking insects such as mosquitos and
sand flies often cause an itchy inflammatory reaction at the site
of the bite. Although the extract mechanism of this reaction is
poorly understood, mast cells and histamine release are likely
components of this reaction (Greaves and Wall, 1996; Horsmanheimo
et al., 1996).
[0296] In preliminary experiments, the inventors treated human sand
fly bites with E. peplus extract and found a rapid reduction in the
itchy sensation compared to untreated bits at a distant site.
Without wishing to be bound by any proposed mechanism, the
inventors believe that the E. peplus extract may strongly stimulate
mast cell exocytosis and histamine release and thereby prevent the
slow release over time of these compounds, a feature associated
with itchiness.
EXAMPLE 15
Promoter Activation as a Means of Therapy: Effect of PEP003 and
PEP004 on Activation of EBV Infected Cell Lines and EBV Positive
Burkitt's Lymphoma Cell Lines
[0297] Initially the effect of PEP003 and PEP004 was tested on the
B95-8 cell line (an EBV positive marmoset cell line that is used
worldwide as one of the best EBV producers). This cell line was
treated with each of these compounds (at different concentrations)
for 3 days and 7 days, respectively, and activation of EBV virus
production was measured by the appearance of a viral capsid antigen
(VCA) on western blots. Also, as a comparison, EBV was activated in
this cell line with TPA.
[0298] To ensure that equal amounts of each sample were analyzed,
the gels were stained with Coomassie blue and the loadings were
adjusted to make them equal. Analyses of VCA in each of the samples
showed that both PEP003 and PEP004 were capable of activating EBV
(at all of the concentrations used) to similar levels as using 65
nM TPA (FIG. 14). Next the PEP003 and PEP004 were assayed on two
Burkitt's lymphoma cell lines and an LCL. This time only
concentrations of 10.sup.-5 and 10.sup.-7 were used. Neither PEP003
and PEP004 had much effect on the LCL (this LCL produces some VCA
without and chemical induction and this was not increased by these
compounds). PEP004 had no effect on VCA production in any of the
cell lines used. However, PEP003 did induce high levels of VCA in
both Burkitt's lymphoma cell lines (MutuI and BL74), but only at
10.sup.5 concentration (FIG. 15). Similar results were obtained
when the cell lines were assayed for induction of BZLF1, the
initial transactivator of EBV replication (FIG. 16). The results
show that PEP003 was capable of activating EBV in Burkitt's
lymphoma cell lines, but appeared to have little effect on
LCLs.
[0299] In conclusion, (1) both TPA and PEP003 can modulate gene
expression in EBV transformed tumor cells at the doses used; (2)
while PEP003 induced VCA in MutuI cells TPA did not, indicating
different modes of action; (3) surprisingly, there was no apparent
effect of PEP003 on lymphoblastoid cells, indicating potential for
activating latent herpesvirus in tumors without affecting the
normal infection.
EXAMPLE 16
Investigation Into the Effect of PEP003 on the Ability of Melanoma
Cells to Stimulate NK Activity
[0300] Melanomas and other cancers can be killed by both specific
(T cell-mediated) and non-specific (natural killer cell and other
mechanisms) arms of the immune response. These killer cells can be
generated in vitro by stimulating peripheral blood T cells from
selected melanoma patients with melanoma cells derived from the
same patient ("autologous"). Natural killer cells can be recognized
by their lysis of the natural killer-sensitive cell line K562. It
has been theorized that some anti-tumor agents alter the
susceptibility of melanomas to immune responses.
[0301] Peripheral blood lymphocytes from patient A02, who has a
strong specific T cell response to her own melanoma cells (A02-M),
were thawed and stimulated by irradiated A02-M pre-treated
overnight at 37.degree. C. with (a) PEP003 (2.25 .mu.g/mL; 50
.mu.M); (b) TPA (100 ng/mL); or (c) control solvent/buffer, and
washed .times.2 before addition to responding lymphocytes (washing
.times. 2 achieves a residual agent dilution of .times. 100,000).
After 10 days of culture, the stimulated cells were harvested and
used as effectors against an NK-sensitive cell line (K562) to test
for the level of NK activity generated in culture. All
determinations were performed in triplicate, at E:T ratios of 45,
15, 5 and 1.7:1. A standard 5 hour.sup.-51 Cr release assay was
performed. Stimulations were performed in 10% fetal bovine
serurn/RPMI-1640.
[0302] The results presented in Table 7 and FIG. 17 indicate that
pre-treatment of melanoma cells with PEP003 significantly increases
the lysis of K562 compared to both TPA and the control treatment at
the E:T ratio of 45:1 (P<0.01 in both cases), suggesting that
PEP003 increases NK activity in A02 cultures.
EXAMPLE 17
Methods for Obtaining a Low-Chlorophyll, Hydrophobic Fraction From
E. peplus and Other Plant Species
[0303] Standard methods for the isolation of hydrophobic compounds
from plants involve alcoholic extraction of the whole plant. This
produces an extract containing chlorophyll and other hydrophobic
substances from the leaves that interfere with subsequent
purification of compounds by solvent extractions and
chromatography. This is a particular problem in isolating highly
bioactive diterpenes from members of the Euphorbiaceae family, due
to co-migration with chlorophyll on silica gel chromatography. Two
methods, both of which can be scaled up for economical, commercial
production, have been developed to overcome this problem, as
described in the present Example and in Example 18.
[0304] Fresh E. peplus plants (17 kg) were chopped and soaked in
150 litres of water at 4.degree. C. for 20 hr. The water was pumped
through 50 and 100 mesh sieves, filtered through 5 and 2 micron
filters and then recirculated through a 100 mm diameter column of
Amberlite XAD-16 (1.5 kg, conditioned successively with ethyl
acetate, methanol and water) at 4.degree. C. (approximately 1.2
L/min) for 72 hr. Adsorption of bioactivity to the resin was found
to be virtually complete within 20 hr.
[0305] The resin was then washed successively with water and 50%
methanol, then eluted with 1 L of methanol, followed by 2.times.1 L
acetone. The eluates were evaporated and combined to give
approximately 7 g of a thick oil. This was shown by HPTLC to be
substantially free of chlorophyll and to contain the desired
ingenane esters which were then purified as described below.
[0306] The ability to extract diterpene esters from chopped plants
in water was surprising given their relative hydrophobicity and
water insolubility. A variety of manual (cutting with scissors) and
mechanical (rotary cutters, motor-driven mulcher) plant maceration
methods were successful, as was extraction at room temperature.
Adsorption to the XAD-16 could be achieved by stirring the resin
with the filtered or unfiltered water extract and then pouring off
the latter. Filtration could also be carried out with minimal loss
of bioactivity using diatomaceous earth, or membrane filters
(220-650 microns). XAD-7 and XAD-4 were as effective as XAD-16.
[0307] The hydrophobic adsorbent polyamide (ICN Biomedical Research
Products) was also used to trap the diterpenes from water; it had
the advantage of allowing the diterpene esters to be selectively
eluted with 50-80% methanol, thus separating them from inactive,
hydrophobic compounds, which remained on the column.
EXAMPLE 18
Method for Separation of Ingenane Esters From Other Diterpenes
[0308] The following method is based upon the surprising discovery
that the stems of E. peplus contain approximately 90% of the
bioactive diterpenes and significantly less chlorophyll compared
with the leaves.
[0309] The plants are dried in air, shaken to remove the leaves and
the stems compressed and covered with an equal weight of methanol
for 24 hr. The solvent is then poured off, evaporated to dryness
under reduced pressure and the residue dissolved in methanol for
chromatography on Sephadex HL20 as described below. This method is
also suitable for isolation of low-chlorophyll fractions from other
plant species.
[0310] A solution of crude methanol extract from E. peplus in 4 mL
90% ethanol was loaded onto a 25 mm.times.1000 mm column and eluted
with 90% methanol. Fractions (4 mL) were analysed by HPTLC (silica
gel, developed with 4:1 toluene: acetone and heated with phosphoric
acid at 110 degrees for 15 min). Typically, fractions 54-63
contained jatrophane and pepluane esters and fractions 64-77 the
ingenane esters, thus achieving satisfactory separation.
Bioactivity, as judged by induction of bipolar morphology in the
human melanoma cell line MM96L, was retained, as for example
disclosed in PCT/AU98/00656.
[0311] This separation was surprising because the polarity of the
ingenane esters as judged by HPTLC on silica completely overlapped
the range shown by the jatrophane and pepluane esters.
EXAMPLE 19
Process for the Purification of Diterpene Esters From E. peplus
[0312] Crude extracts obtained by the methods according to Examples
17 or 18 above, or by ether extraction of latex, were fractionated
by Sephadex HL-20 chromatography (as above). Appropriate fractions
from the latter were combined, the methanol evaporated under
reduced pressure and the remaining water removed by freeze-drying
or by ether extraction. This sample (200 .mu.L of 100 mg/mL in
methanol per injection) was fractionated by HPLC on a Phenomenex
Luna 250.times.10 mm C18 column with a Phenomenex guard column in
70-100% methanol at 2 mL/min, with detection at 230 nm. Jatrophane
and pepluane esters appeared at 25-42 min, PEP005 at 42-44 min,
PEP008 at 46-50 min, and PEP006 at 50-54 min. Similar types of
separation have been obtained by HPLC on C3 and C8 columns.
[0313] Fractions pooled from repeated runs were evaporated to
dryness (rotary evaporater or freeze dryer), and stored in acetone
at -20.degree. C. under argon or nitrogen.
EXAMPLE 20
Activation of Leukocytes by Diterpene Esters, for Selective Killing
of Human Tumor Cells in Culture
[0314] Leukocytes obtained by lysis of human peripheral blood were
added to 5000 MM96L human melanoma cells or 7000 neonatal foreskin
fibroblasts per microtitre well at effector: target ratios of 1000,
100 and 10:1. Ing9 (60 ng/mL) was added and after 48 hr incubation
the cultures were washed and labelled with [3H]-thymidine for 2 hr.
At 100:1 ratio of effector:target cells, the melanoma cells showed
9% survival with PEP008 whereas the normal fibroblasts had 100%
survival. Untreated leukocytes had no effect on cell survival.
[0315] These results indicate that the diterpene esters of the
invention activate human peripheral blood leukocytes to produce, in
a PKC-dependent manner, phagocytosis and a respiratory burst which
are potentially lethal to micro-organisms and other cells.
[0316] This example shows that drug-activated, PKC-dependent
processes can direct tumor-specific killing by cells of the innate
immune system.
EXAMPLE 21
Pretreatment of Human Tumor Cells in Culture With Diterpene Esters
Potentiates Selective Killing by Untreated Leukocytes
[0317] The question of whether drug treatment of the target tumor
cells causes them to become susceptible to effector cells of the
immune system was addressed as follows.
[0318] Leukocytes obtained by lysis of human peripheral blood were
added to 5000 MM96L human melanoma cells or 7000 neonatal foreskin
fibroblasts per microtitre well at effector: target ratios of 1000,
100 and 10:1. The target cells had been treated with 60 ng/mL
PEP008 for 20 hr beforehand, and washed and the medium replaced
before the leukocytes were added. After 48 hr incubation with the
leukocytes the cultures were washed and labelled with
[3H]-thymidine for 2 hr. At 100:1 ratio of effector:target cells,
the melanoma cells showed 12% survival with PEP008 whereas the
normal fibroblasts had 100% survival. Untreated leukocytes had no
effect on cell survival.
[0319] This result showed that the drugs also act by making tumor
cells specifically sensitive to lysis by the immune system.
EXAMPLE 22
Topical Composition A for the Treatment of Conditions Affecting
Skin (e.g. Infections, Skin Cancers)
[0320] Tinctures: Compounds of the invention were diluted into
acetone, ethanol or isopropanol to the same final bioactivity as
the E. peplus latex as measured by bipolar activity in MM96L human
melanoma cells (10 million bp units per mL). Samples (2-5 .mu.L)
were applied daily for 3 days to the surface of mouse melanoma B16
tumor 3-5 days after implanting s.c. 1 million cells on the flanks
of nude mice. Efficacy, defined as 67% or more sites cured, was
obtained for E. peplus sap, PEP005, PEP008 and a mixture of PEP005,
PEP006 and PEP008.
EXAMPLE 23
Topical Composition B for the Treatment of Conditions Affecting
Skin (e.g. Infections, Skin Cancers)
[0321] Creams and gels: A variety of hydrophobic cream bases was
found to be ineffective when used to deliver compounds to the skin
as described above for the tinctures.
[0322] Efficacy was obtained with the use of an isopropanol gel,
formulated as described for the tinctures.
[0323] The results show that E. peplus sap and its terpenoid
components activate PKC, with consequent potential to induce a wide
range of cellular responses without the high tumor promoting
activity of TPA. The carboxypeptidase activity may have application
in enhancement of tissue penetration and in antigen processing for
optimal immune responses.
[0324] Overall, the results indicate that E. peplus extract induces
a set of cellular responses with affects PKC, cell cycle genes and
inflammatory mediators, some but by no means all of which are
similar to the action of TPA. In particular, the results indicate
that E. peplus sap and its terpenoid components are useful in the
treatment of a variety of infections and as adjuvants for
stimulating immune responses.
EXAMPLE 24
Effect of Saps Derived From Other Members of the Euphorbiaceae
Family on MM96L Cells
[0325] Sap was collected from Synadenium grantii, Synadenium
compactum, Mondenium lugardae, Mondenium guentheri, Endadenium
gossweileni, and E. peplus and serially diluted ten-fold up to
10.sup.-7into sterile 1.5 mL Eppendorf.TM. tubes using growth
medium. Ten-microlitre aliquots of each dilution, in the presence
or absence of the PKC inhibitor bisindolylmaleimide (1 .mu.g/mL or
10 .mu.g/mL), were added to 5000 MM96L cells per well of a
microtitre plate. After 3 days, cells were examined for
cytotoxicity or differentiation to a bipolar dendritic
phenotype.
[0326] The results presented in Table 8 show that the saps of S.
grantii, S. compactum, M. lugardae, M. guentheri, and E.
gossweileni, like that of E. peplus, induce the differentiation of
MM96L cells to a bipolar phenotype and that this differentiation is
inhibited by the bisindolylmaleimide. This inhibition strongly
suggests that the active components of the saps induce cell
differentiation by inhibition of PKC activity. The results also
show that at higher concentrations (10.sup.-4 and above), the saps
are effective in killing MM96L cells.
EXAMPLE 25
Effect of Saps Derived From Other Members of the Euphorbiaceae
Family on JAM Cells
[0327] The saps of Example 24 were also examined for their
cytotoxic effect on the ovarian carcinoma cell line JAM.
Ten-microlitre aliquots of each dilution of sap, prepared according
to Example 24 in the presence or absence of the PKC inhibitor
bisindolylmaleimide (10 .mu.g/mL), or in the presence or absence of
the PKC phorbol ester binding site ligand phorbol dibutyrate, were
added to 5000 JAM cells per well of a microtitre plate. After three
days, the cells were fixed with ethanol and the number of cells
compared with untreated controls stained with sulfurhodamine B.
[0328] The results presented in FIGS. 18A and 18B indicate that,
like the sap of E. peplus, the saps of S. grantii, S. compactum, M.
lugardae, M. guentheri, and E. gossweileni, at concentrations of
10.sup.-4 and above, are effective in killing JAM cells. These
results also show that cytotoxicity is inhibited by
bisindolylmaleimide, suggesting that this effect is mediated by
modulation of PKC.
[0329] Inspection of FIG. 18C reveals that the cytotoxic effects of
saps derived from M. guentheri and E. gossweileni were blocked in
the presence of phorbol dibutyrate, suggesting that the active
components of these saps mediate their cytotoxicity by binding to
the phorbol ester binding site of PKC.
[0330] Those skilled in the art will appreciate that the invention
described herein is susceptible to variations and modifications
other than those specifically described. It is to be understood
that the invention includes all such variations and modifications.
The invention also includes all of the steps, features,
compositions and compounds referred to or indicated in this
specification, individually or collectively, and any and all
combinations of any two or more of said steps or features.
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* * * * *
References