Note: Descriptions are shown in the official language in which they were submitted.
WO 95/14466 21 l 6 6 0 3 pcT/US94l13260
NOVEL PALLADIUM COMPLEXES AND METHODS FOR
USING SAME IN THE TREATMENT OF TUMORS AND PSORIASIS
$ACKGROUND OF THE INVENTrnN
1. Field of the Invention
The present invention relates generally to novel palladium complexes
and pharmaceutical compositions comprising same. In addition, a method
for the preparation of novel palladium complexes is disclosed. Also
disclosed is a method for the treatment of tumors comprising administering
the novel palladium complexes of the present invention and a method for the
treatment of psoriasis comprising administering the novel palladium
complexes of the present invention.
2. Related Art
A number of researchers have studied the role that nucleic acids play
in tumorigenesis. Although electron reduction of single nucleotides have
been reported in, for example, Reichard, "From RNA to DNA, Why So
Many Ribonucleotide Reductases?" i nc 260:1773-1777 (June 18, 1993);
and Hamilton et al., "Cobamides and Ribonucleotide Reduction VII
Cobalt(II)alamin as a Sensitive Probe for the Active Center of
Ribonucleotide Reductase," Biochemistry 10(2):347-355 (1971), previous
reviews of nucleic acids and their role in tumorigenesis make no mention of
reactions or substances which result in the electron reduction of DNA or
RNA. Townsend et al. (ed), Nucleic Acid hemistrv Part 3, Wiley
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WO 95/14466 2 ~ 7 6 ~ Q 3 PCTIUS94/13260
Interscience, New York (1986); Saenger, Principles of Nucleic Acid
ru tur , Springer-Verlag, New York (1984); Walker, "Nucleic Acids,"
Methods in Molecular Biology, vol. 2, Humana Press, Clifton, NJ (1984);
Adams et al., ~'he Biochemistry of the Nucleic Acids, part 3, Wiley
Interscience, New York (1986); Grossman et al. (ed), "Nucleic Acids - part
1," Methods in Enz, molog~t, vol. 65, Academic Press, New York (1980);
Fasman (ed), "Nucleic Acids," ~Iandbook of Biochemistry and Molecular
BioloQV (3d), vol. 1, CRC Press, Cleveland, OH (I975); Blackburn et al.
(ed), Nucleic Acids in Chemistry and BioIo,.gY, IRL Press, Oxford Univ.
Press (1990):
The prevailing view of tumorigenesis today is that it may be treated
by site-specific regulation, such as by a repressor protein, at proto-oncogene
sites. Such proto-oncogene sites are different for each type of tumor being
treated and an extensive effort is required in order to pursue this method of
treatment for an individual patient.
In 1969 McMuIlen described a model of the electrostatic free energy
of nucleic acids. McMullen, "The Electrostatic Free Energy of
Macromolecular Systems," Electronic Aspects of Biochemistry Annals NY
Acad. Sci., Vol. 158, Art. 1, 223-239 (May 1969). Purugganan et al. in
1988 described the electron energy interactions of DNA. Purugganan et al.,
"Accelerated Electron Transfer Between Metal Complexes Mediated by '
DNA," cience 241:1645-1649 (Sept. 23, 1988). ,
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WO 95114466 ~ ~ ~ PCT/US94113260
The first redox regulation of the transcription of the proto-oncogenes
c-fos and c jun was reported in 1990 in a landmark paper by Abate et aL,
"Redox Regulation of Fos and Jun DNA-Binding Activity in Vitro," cience
249:1157-1161 (September 7, 1990). Abate et al. had previously identified a
nuclear factor that stimulates the DNA-binding activity of fos and jun in
vitro. This factor did not bind to the fos-jun complex or to the DNA
regulatory element known as the activator protein-1 (AP-1) binding site,
thereby suggesting that it regulated DNA-binding activity indirectly. The
authors hypothesized that the nuclear factor reduces a critical cysteine
residue in fos and jun that is required for DNA-binding activity. It was
believed that one or more cysteine residues in fos and jun are important for
DNA binding and that reduction is required for association with DNA.
Abate et al. at 1158. The findings of Abate et al. thus suggested that
modification of the redox state of fos and jun may contribute to the
formation of specific protein-DNA complexes. The bacterial transcriptional
regulatory protein, Oxy R, which regulates gene expression in response to
oxidative stress, was found to change DNA-binding specificity depending on
the redox state. Thus, regulation by reduction-oxidation was believed to be
a mechanism of control for certain transcription factors.
Recently, Shaw et ai. studied the free energy of formation of relaxed
. trefoil and figure-eight DNA knots. Supercoiled trefoil DNA knots were
also evaluated. The authors found that the presence of a knot in a relaxed or
supercoiled DNA ring is associated with a substantial free energy cost.
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WO 95/14466 _ PCT/US94/13260
2176603
Furthermore, in enzyme-catalyzed reactions that yield
knotted products, this free energy cost must be
compensated for by a favorable free energy term, such as
that derived from protein-DNA interactions. Shaw et al.,
"Knotting of a DNA Chain During Ring Closures," Science
260:533-536 (April 23, 1993).
In spite of the above described research relating to
tumorigenesis to date, the conventional methods for the
treatment of tumors in individual patients have not met
with resounding success. Thus, the search for a new
method or methods for the treatment of tumors continues.
Likewise, there are other disease states and conditions,
such as psoriasis, which have long sought a safe and
effective method of treatment.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows crystallographic studies of the Pd-
lipoic acid complex. The figure shows the trigonal prism
of palladium-lipoic acid complex (Poly-RC).
Figure 2A shows an ultraviolet spectra of
uncomplexed sodium lipoate, aqueous 10'4 molar (experiment
conducted 6/11/93). There is no UV peak. Figures 2B and
2C show ultraviolet spectra of Pd-Lipoic Acid Complex
4 '
RECTIFIED SNEET (RULE 91)
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WO 95114466
PCTIUS94113260
(Poly-RC) with a concentration of 2x10-6 M in Figure 2B and 10-6 M in Figure
2C.
Figures 2D and 2E show ultraviolet absorbance spectra of oxidized Poly RC
after
ninety days of air oxidation of the solution (experiment conducted 6/1//93).
In
Figures 2D and 2E, the concentration is .00012 M. In Figure 2D, the spectrum
is
ended at 400.0 nm and 0.542 A, and a reference peak is at 289.0 nm and 0.75 A.
In Figure 2E, the spectrum is ended at 550.0 nm and 0.036 A, and a reference
peak
is at 396.0 nm and 0.553 A. Figure 2F illustrates the absorbance spectra of Pd-
lipoic acid complexes at varying ratios of palladium to lipoic acid at a Pd
concentration of 5 x10-5 M. The ratios are 1 Pd : 1 lipoic = n 1, 1 Pd : 2
lipoic = n 2,
and 1 Pd : 3 lipoic = n 3. Figure 2 G illustrates an ultraviolet spectra of
Poly-R2
(Polynucleotide reductase - 2)(concentration 2 x 10-6 M) at frequencies
between 195
and 215 nm. Figure 2H shows an ultraviolet spectra of bis (thiamine-HCI) Pd at
a
concetnration of 5 x 10-" M. Figure 21 illustrates a comparsion of the
ultraviolet
spectra of vitamin B,z with the activated derivative of vitamin B,z at a
concentration of
1.5 x 10-6 M in an experiment conducted 5/12/93. The reference peak of B,2 is
at
361.2 nm and 0.042 A and the reference peak of B,2Ac is at 358.6 nm abd 0.044
A.
25
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WO 95/14466 PCT/US94113260
217663
Figure 3A shows an FTIR scan of Pd-lipoic acid in
KBr Mull in an experiment dated 17 Dec 92. Figure 3B
shows an FTIR scan of Pd-lipoic acid complex in KBr Mull
an experiment dated 17 Dec 92. Figure 3C compares the
FTIR scan of Pd-lipoic acid complex and that of lipoic
acid in experiments dated 17 Dec 92.
In Figures 4A to 7B, the scan direction is (-+) and
the scan rate is 100mV/SEC.
Figures 4A, 4B, 4C and 4D illustrate cyclic
voltammetric scans of Poly-RC and Poly-R2. Figure 4A
shows the electrochemical signature of a solution of Poly
RC in acetate buffer, PLC: 250 x,1/.12 M, in a scan from 0
to -1.0 volts at 20 ~.A sensitivity in an experiment dated
2-23-93. Figure 4B shows a solution of .3 ml Poly-R2
( . 08 M) in scan from 0 to -1 . 0 volts at 100 ACA
sensitivity in an experiment dated 3-16-93. Figure 4C
shows a solution of .3 ml Poly-R2 (.08 M) in scan from
.3 to -1.0 volts at 20 ~.A sensitivity in an experiment
dated 3-16-93. Figure 4D is a comparison of signals of
Poly-RC and Poly-R2. Figures 4E and 4F illustrate cyclic
voltammetric scans of the activated Bi2-Poly-R2 complex.
Figure 4E shows the signature of B~ZAc-Poly-R2 in acetate
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RECTIFIED SHEET (RULE 91)
WO 95/14466 PCT/US94/13260
buffer solution .6 ml/.04 M in a scan from -.3 to -1.0
volts at 20 ACA sensitivity in an experiment dated 5-19-
93. Figure 4F shows the signature of B~ZAc-Poly R2 in
acetate buffer solution, .6 ml/.04 M in a scan from 0 to
a
-1.0 volts at 20 ~,A sensitivity in an experiment dated
5-19-93.
Figure 5 shows the charge interaction of Poly-RC in
a solution of 250 x,1/.12 M plus DNA (CT) in a solution of
1.0 ml of 5 mg/ml in a scan from 0 to -1.0 volts at 20 ~.A
sensitivity in an experiment dated 2-23-93. Figure 5
shows DNA oxidizes Poly-RC.
Figures 6A and 6B illustrate cyclic voltammetry
patterns for Poly-R2 interaction with DNA. Figure 6A
shows a solution of .3 ml Poly R2 (.08 M) plus 1.0 ml DNA
5 mg/ml in a scan from 0 to -1.0 volts at 100 ACA
sensitivity in an experiment dated 3-16-93. Figure 6A
shows DNA oxidizes Poly-R2. Figure 6B shows an acetate
buffer solution of .3 ml Poly-R2 (.08 M) plus 1.0 ml DNA
(CT) 5 mg/ml in a scan from -.3 to -1.0 volts at 20 ACA
sensitivity in an experiment dated 3-18-93. Figure 6B
shows DNA oxidizes Poly-R2. Figure 6C shows a cyclic
voltammetry pattern of the reaction of Poly-R2 and yeast
RNA. Figure 6C shows a solution of Poly-R2 plus RNA in a
5-3
REC1~F~EU SHEET (RULE 91)
WO 95/14466 PCT/ITS94/13260
217~~~~3
scan from -.3 to -1.0 volts at 20 ACA sensitivity in an
experiment dated 3-30-93.
Figure 7A shows cyclic voltammetry patterns of the
interactions of Poly-R2, DNA and vitamin B~2. Figure 7A
4
shows a solution of Poly-R2 (.3 ml: .08 M) plus DNA (1.0
ml: 5 mg/ml) plus BIZ (1.0 ml: .2 mg/ml) in a scan from -
.3 to -1.0 volts at 20 ACA sensitivity in an experiment
dated 3-31-93. Figure 7A shows BIZ induces a second and
further oxidation of Poly-R2 after the DNA. Figure 7B
illustrates a cyclic voltammetry pattern of the reaction
of the activated Bi2-Poly-R2 complex and DNA. Figure 7B
shows a solution of (1) B~ZAC-Poly R (.6 ml/.04 M) and (2)
DNA (CT) .5 ml (5 mg/ml) in a scan from -.3 to -1.0 at 20
~A sensitivity in an experiment dated 5-19-93. Figure 7B
shows rapid achievement of equilibrium.
Figure 8A illustrates induction of giant forms of
Baker's yeast by DNA reductase and altered cell
morphology of yeast cells to which Poly-R2 was added.
Figure 8B shows a stage in the giantization of the yeast
cells in which lipid drops are lost from the cell.
Figure 9 shows the effect of B~2 and Poly-R2 on
Baker's yeast cells showing cell enlargement with
heterochromatin formation.
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WO 95/14466 PCT/US94l13260
2l 76603
Figures 10A, lOB, lOC, 11A, 11B, 12A, 12B, 12C, 13A
' and 13B are clinical photographs taken of patients being
treated for psoriasis with B~2AC-Poly-R2.
Figure l0A shows R anterolateral abdomen with
psoriasis to compare treated areas (R side of photo) with
untreated areas (L side of photo). R side has been
treated with Polyredox applied once daily for 7 days.
Psoriatic lesions appear less scaly, less thick and
smooth stretch marks of surrounding and underlying skin
are more visible through thinner plaques. Untreated
areas on L side remain with thick, scaly, psoriatic
lesions. Figure lOB shows an untreated psoriatic plaque,
close up, showing thick, silvery scales with underlying
red inflamed base. Figure 10C shows an area of abdomen
treated with Polyredox applied once daily for 7 days.
Already there is less scaling and obvious smoothening of
lesions. Normal skin markings are visible through the
thinner plaques. Redness is from the stain of the
compound.
Figure 11A shows L lateral thigh with psoriasis
before treatment: thick papulosquamous plaques showing
silvery scales and red inflamed base. Figure 11B shows L
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RECTIFIED SHEET (RULE 9ij
WO 95/14466 PCT/US94/13260
lateral thigh with psoriasis area on L side of photo
treated with Polyredox twice daily for 7 days. Treated
area appears smoother and thinner.
Figure 12A shows L lateral thigh with psoriatic
plaque before treatment. Figure 12B shows the same
lesion treated with Polyredox twice daily for 2 weeks.
Lesion appears flatter and contracted centrally with
overall diminution in size. There is overlying yellowish
"scab" giving an impression of dryness. Superficial
fissuring may signify the drying effect and/or
contraction of sections of the lesion. Smaller lesion at
2 o'clock remains unchanged. Figure 12C shows the same
as Figure 12B at higher magnification. Features
mentioned above appear more obvious. Small lesion at 10
o'clock is superficially eroded and appears necrotic.
Figure 13A shows forearms, dorsolateral view,
encased in thick plaques of psoriasis prior to treatment
with Polyredox. ,Figure 13B shows the same forearms one
week later. The L forearm was treated with Polyredox
applied 2x daily for 7 days. The R forearm is untreated.
Treated areas on the L forearm show less scaling,
smoothening and thinning of lesions. Improvement is
specially noted on the area below the L elbow (compare
with Figure 13A).
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WO 95114466 PCT/US94/13260
OB.TECTS AND SUIYllVIARY OF THE INVENTION
The present inventor has taken a different approach to the treatment
of tumorigenesis from those of the prior art. Surprisingly, it has been
discovered that by altering the enzymatic or catalytic pathway represented by
even a singular or very few gene sites, the native conformation of large
tracts of DNA can be altered. The present inventor believes that electron
energy from a normal metabolic hydrogen carrier, such as lipoic acid, can be
shunted to nucleic acids. The electron energy which is shunted can be
measured by conventional voltammetric means.
An object of the present invention is to provide a novel
polynucleotide reductase which is a complex comprising palladium or a salt
thereof, and lipoic acid or a derivative thereof. The complex may comprise
the formula (palladium)m(lipoic acid)n wherein m and n are each
independently 1 or 2.
It is also an object of the present invention to provide a complex for
administration to a patient wherein the palladium-Iipoic acid complex is
present in an amount sufficient to obtain a concentration of at least about
0.01 M. The palladium-lipoic acid complex is preferably present in a
dispersion or solution.
It is a further object of the present invention to provide a thiamine
salt of a palladium-lipoic acid complex composition wherein the palladium,
lipoic acid and thiamine are present in a solution at a pH such that a
complex is formed.
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WO 95/14466
PCT/US94/13260
It is a still further object of the present invention to provide a
complex comprising palladium-lipoic acid, which further comprises the
addition of other ligands to the palladium-lipoic acid complex.
Another object of the present invention is to provide a thiamine salt
of a palladium-lipoic acid complex which further comprises a synthetic
cofactor of vitamin B12 (cyanocobalamin).
Additionally, the present im~ention relates to a method of synthesizing
a palladium-lipoic acid complex.
It is a further object of the present invention to provide a
pharmaceutical composition of matter comprising a pharmaceutically
effective amount of a complex of palladium or a salt thereof to treat a tumor
or psoriasis, and lipoic acid or a derivative thereof, and a pharmaceutically
acceptable carrier therefor. Further, a pharmaceutical composition of matter
comprising a palladium-lipoic acid complex which also comprises thiamine
or a salt thereof is provided. In addition, a pharmaceutical composition of
matter comprising a palladium-Iipoic acid-thiamine complex which further
comprises as a synthetic cofactor vitamin B12 (cyanocobalamin) is provided.
The present invention also relates to a method of treatment of tumors
comprising administering the pharmaceutical composition of matter
comprising a complex of palladium or a salt thereof and Iipoic acid or a
derivative thereof in an amount effective for tumor reduction to a patient, '
e.g., human, in need of such treatment. A further method of treatment of
tumors comprises administering a palladium-lipoic acid complex which also
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WO 95/14466 PCT/US94I13260
comprises thiamine or a salt thereof in an amount effective for tumor
reduction to a patient in need of such treatment. In addition, a method of
treatment of tumors comprising administering a palladium-Iipoic acid-
thiamine complex which comprises a synthetic cofactor of vitamin B12
(cyanocobalamin) in an amount effective for tumor reduction to a patient in
need of such treatment is provided.
In addition, the present invention relates to a method of treatment of
psoriasis comprising administering the pharmaceutical composition of matter
comprising a complex of palladium or a salt thereof and lipoic acid or a
derivative thereof in an amount effective for reduction of maculopapules
associated with psoriasis to a patient in need of such treatment. A further
method of treatment of psoriasis comprises administering a palladium-Iipoic
acid complex which also comprises thiamine or a salt thereof in an amount
effective for reduction of 'maculopapules associated with psoriasis to a
patient
in need of such treatment. In addition, a method of treatment of psoriasis
comprising administering a palladium-Iipoic acid-thiamine complex which
comprises a synthetic cofactor of vitamin B12 (cyanocobalamin) in an amount
effective for reduction of maculopapules associated with psoriasis to a
patient
in need of such treatment is provided.
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PCTIUS94113260
DESCRIPTION OF THE PREFERRED EMBODIMENTS
As previously stated, the present invention relates to the transfer of
electron
energy from a normal metabolic hydrogen carrier to nucleic acids. As used in
the
present specification, the term "polynucleotide reductase" is a compound which
is
capable of shunting electron energy from itself to nucleic acids. In the
terminology
of organic chemistry, the reductases are "sources" which are able to donate
electrons into the nucleic acid "sinks". Scudder, Electron Flow if Organic
Chemistry,
John Wiley, 2-3 (1992). DNA has previously been described as in intermediate
for
electron transfer reactions in Purugganan et al., "Accelerated Electron
Transfer
Between Metal Complexes Mediated by DNA" Science 241: 1645-1649 (Sept. 23,
1988)", which reported that a double-stranded DNA polymer can mediate long-
range
electron transfer between bound donor-acceptor pairs.
The present inventor has found that a palladium-lipoic acid complex of the
present invention can function as a polynucleotide reductase to transfer
electrons
into DNA and RNA. Further, and without wishing to be bound by any theory, the
present inventor believes that when the electron energy from a palladium-
lipoic acid
complex of the present invention is shunted to DNA or RNA, it alters the
nucleic acid
configuration. A polunucleotide reductase capable of shunting electron energy
from
itself to DNA is termed a DNA reductase, while a polynucleotide reductase
capable
of shunting electron energy from itself to RNA is termed a RNA reductase.
The present inventor has discovered a novel palladium-lipoic acid complex.
The complex may exist in a solid form, however, the complex is
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WO 95/14466 PCTIUS94I13260
preferably in a liquid form as a dispersion, or more preferably as a solution.
The complex, also referred to as a coordination compound herein, is a
compound containing a metal atom or ion bonded by at least one ionic bond
to a number of anions or molecules. The complexes of the present invention
comprising a transition metal ion are thermodynamically stable.
As is common.in metal to ligand syntheses, multiple complexes of
palladium with lipoic acid may be produced. The general formula of the
complex of the present invention is (palladium)m(Iipoic acid)n, wherein m
and n are each independently 1 or 2. Preferably, m is l and n is 1. At least
two forms of the Pd-lipoic acid complex have been identified by
spectroscopy. These two forms of the Pd-lipoic acid complex are as
follows: (I) a 1:1 (n=1) ratio of palladium to lipoic acid, and (2) a 1:2
(n=2) ratio of palladium to lipoic acid. The structure of each of these
complexes is as follows:
H C~CH2~ H
2 C
CH2 n a 1
CH
\ S CH2
2
' \Pd
/ \I~I CH2
C
WO 95/14466 2 ~ l 6 ~ ~~ ~ PCT/US94/13260
/CH2~ H
H2C C~CH2
~CH2v
S S
' \Pd - CH2~CH2v
C=O
I
O~C O O_
~ H2
CH2
CH2
/CH2-CHI
CH2 S n = 2
~H2C-S~
The bonds of the palladium-lipoic acid complex are coordinate
covalent. More specifically, studies have shown that the palladium-lipoic
complex is bonded by coordinate covalent bonds: (1) at the carbonyl end of
the substituent having a carboxyl group with probable resonance involvement
of both oxygens, and (2) at one or more sulfur atoms. The lipoic acid in the
complex comprises a bent carbon chain with the ends of the chain bonded to
the palladium. This 1:1 structure is represented above as a bent cyclic
structure. However, while the figure shows a planar structure,
crystallographic studies as discussed below show the structure to be three-
dimensional with the palladium in the center of the complex.
As previously stated, Iipoic acid is one component of the complex of
the present invention. The present inventor has found that Iipoic acid and its
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WO 95/14466 ~ ~ 7 6 6 0 3 pCTJUS94/13260
derivatives are highly specific for transferring electron energy from a normal
metabolic hydrogen carrier to nucleic acids. Lipoic acid occurs in an
oxidized or disulfide form, or in a reduced or dithiol form. The structure of
lipoic acid in its oxidized form is as follows:
H2C/CH2~C~H O
\S-S~ 'CH2 CH2 CH2 CH2 C\
OH
Lipoic acid
The structure ~of the reduced form of lipoic acid, i.e., dihydrolipoic acid,
is
as follows:
/CH2~ H
O
HZC~S S C~CH2-CHI-CH -CH - ~~
H H 2 2 C
OH
Lipoic acid (reduced)
As can be seen by the above structures, Iipoic acid has a long,
flexible side chain, which enables it to rotate from one active site to
another
in enzyme complexes. As shown in Campbell et al., Biochemi trv
llus t , 2d, Churchill Livingstone, 126 (1988), lipoic acid is a hydrogen
carrier and an acetyl-group carrier for the decarboxylation of pyruvic acid.
Lipoic acid is then present as acetyllipoic acid, having both an acetyl group
12
CA 02176603 2004-03-04
and a hydrogen atom. In the pyruvic decarboxylation reaction, the acetyl group
is
donated to CoA and the H is donated to NAD+.
In addition, the palladium-lipoic acid complex of the present invention may
further comprise at least one ligand to the palladium-lipoic acid complex. For
example, the additional ligand to the palladium-lipoic acid complex may be an
iorganic anionic ligand, including without limitation acetate,
acetylacetonate, amine,
ammonium chloride, ammonium nitrate, bromide, chloride, fluoride, iodide,
nitrate,
nitrite, oxalate, oxide, pyridine, sulfate and sulfide. The lipoic acid
derivative may
further comprise additional cations, for example, sodium, potassium,
magnesium,
calcium, ammonia, vanadate, molybdate, zinc and tin. Furthermore, the lipoic
acid
of the complex of the present invention may be present in its
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WO 95/14466 , PCTlUS94113260
reduced or oxidized form. Other derivatives of Iipoic acid known in the art
may also be used in the practice of the present invention: The derivatives
are suitable if the ability to transfer electron energy from a normal hydrogen
carrier to a nucleic acid is retained. As used in the present specification,
the
term "lipoic acid" is intended to include the derivatives specifically
identified
supra as well as other derivatives known in the art. The features of lipoic
acid believed to be necessary for the present invention include at least two
sulfur atoms, a hydrocarbon chain having a length of two to twenty carbon
atoms, and one or more carboxyl groups.
The metal ion of the novel complex of the present invention is
palladium. Palladium is a transition metal of Group VIII of the periodic
table. Salts of palladium may also be employed in preparing the Pd-lipoic
acid complexes of the present invention. The palladium salts may be
selected from, and are not limited to, for example, palladium acetate,
palladium acetylacetonate, palladium ammonium chloride, palladium
ammonium nitrate, palladium bromide, palladium chloride, palladium
diamine nitrite, palladium diamylamine nitrite, palladium dibromide,
palladium difluoride, palladium dioxide, palladium dipyridine nitrite,
palladium ethylenediamine nitrite, palladium iodide, palladium monoxide,
palladium nitrate, palladium oxalate, palladium oxide, palladium sulfate,
palladium sulfide, palladium tetramine dichloride, palladous potassium
bromide, palladous potassium chloride, palladous sodium bromide, and
palladous sodium chloride. The preferred palladium salts are palladium
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WO 95114466 217 b 6 ~ 3 PC~'IUS94/13260
chloride, palladium bromide, palladium iodide, palladium nitrate, palladium
oxide and palladium sulfide. The most preferred palladium salt is palladium
chloride.
While palladium or a salt thereof is required in the practice of the
present invention, the complex may also further comprise an additional metal
compound such as vanadate, molybdate, zinc or tin, or other canons such as
potassium or sodium.
As discussed supra, at least two forms of the Pd-Iipoic acid complex
are useful in the practice of the present invention:
[1] (palladium)m(Iipoic acid)n wherein m and n are both 1 (i.e., the 1:1
complex), and [2] (palladium)m(lipoic acid)n wherein m is 1 and n is 2 (i.e.,
the 1:2 complex). Other possible forms of the Pd-lipoic acid complex
include (palladium)m(Iipoic acid)n wherein m is 2 and n is 1, and
(palladium)m(lipoic acid)n wherein m and n are both 2. With respect to the
L:1 and 1:2 complexes, studies have shown that the 1:2 complex has less
biological activity than the 1:1 complex. While not wishing to be bound by
any specific theory, the foregoing suggests that the second lipoic acid
molecule competes with the palladium for interaction at the biological site.
With respect to the various palladium-lipoic acid complexes possible, the
equilibria of which complex is favored may be controlled by controlling the
amount of lipoic acid introduced into the reaction.
Crystallographic studies, as illustrated in Figure 1, show that the
palladium-Iipoic acid complex forms a trigonal prism. Trigonal prism
WO 95/14466 217 6 6 0 3 p~/Ug94/13260
symmetry is representative of an octa-coordinate molecule, as stated in
Cotton et aL, Advanced Inorgar,;~ rhemistrv, Interscience, 29 (1972). The
crystallographic studies as illustrated in Figure 1 are thus consistent with
the
prior illustrations of the palladium-lipoic acid complexes set forth above
which show octa-coordination.
Other Pd-lipoic acid complexes are also possible. For example, a
Binuclear complex satisfies octa-coordination. In such a complex, two metal
atoms share the center of symmetry of the complex with four coordinate
covalent bonds between the Iipoic acid molecules and the palladium ions.
The four coordinate covalent bonds could be satisfied, for example, by
having between two to four lipoic acid molecules bonded to the palladium
ions. For example, a Binuclear 2(n=1) complex, wherein two palladium
ions are present with each having one Iipoic acid molecule bonded to it by
two coordinate covalent bonds, could exist. Alternatively, the Binuclear
complex could be a 2(n=2) complex, wherein two palladium ions are
present with each having two lipoic acid molecules bonded to it. In this
Binuclear complex, each lipoic acid is bonded by only one coordinate
covalent bond to each palladium ion. In the present invention, the Binuclear
2(n=1) complex is preferred.
Oxidized and reduced forms of the palladium-Iipoic acid complex are
also contemplated. Whether the oxidized or reduced form is favored will
depend upon the pH of the particular solution containing the Pd-lipoic acid
complex.
I6
WO 95/14466 PCT/US94/13260
2176603
The palladium-lipoic acid complex of the present invention may be
produced by dissolving lipoic acid in a basic solution and adding that to an
acidic solution containing palladium or a salt thereof. The resulting solution
is heated to a boil, e.g., to about 100°C, to produce the Pd-lipoic
acid
complex. More specifically, the palladium-lipoic acid complex of the
present invention may be synthesized by the following procedure:
(a) adding palladium or a salt thereof to an acidic solution;
(b) heating the palladium-acidic solution to at least about 100°C;
(c) filtering the palladium-acidic solution from step (b);
(d) ~dissolving lipoic acid in a basic solution;
(e) adding the dissolved lipoic acid solution from step (d) to the
palladium solution from step (c); and
(f) heating the lipoic acid-palladium solution to at least about
100°C, for an amount of time sufficient to obtain a palladium-lipoic
acid
IS complex.
The palladium or salt thereof is added to the acidic solution in a mole
ratio of between about 1 and about 2 moles palladium to moles acid. Any
method for mixing the palladium and acidic solution may be used, for
example, stirring or agitation. The palladium-acidic solution may then be
heated to a gentle boil, e.g., at least about 100°C, preferably between
about
100° and about 200°C, and most preferably at about 100°C.
The acidic solution to which the palladium is added is selected from
acids well known in the art. Such acids include perchloric acid, sulfuric
17
WO 95/14466
PCT/US94/13260
acid, hydroiodic acid, hydrobromic acid, hydrochloric acid, nitric acid,
phosphoric acid, nitrous acid, acetic acid, carbonic acid, and hydrogen
sulfide. Preferably, the acidic solution is hydrochloric acid.
The palladium-acidic solution may be fltered by any method
generally known in the art. Such methods include, for example, gravity
filtration, suction filtration, centrifugation or the Like.
In a separate container, Iipoic acid is added to the basic solution in a
mole ratio of between about 1 and about 7 moles lipoic acid to mole base.
Any method for mixing the lipoic acid-basic solution may be used, for
example, stirring or agitation. Any of the above methods of filtration may
then be used to eliminate any undissolved residue. The solution is preferably
filtered to complete clarity.
The basic solution in which the Iipoic acid is dissolved can be
selected from bases known in the art, for example, sodium hydroxide,
ethanolamine, potassium hydroxide, sodium acetate, dimethyl amine, diethyl
ether, ethanol and the like. Preferably, the basic solution is sodium
hydroxide.
Next, the dissolved lipoic acid solution is added to the palladium
solution. The palladium-Iipoic acid solution is heated to a gentle boil, e.g.,
to at least about 100 °C, preferably to between about 100° and
about 200°C,
and most preferably to about 100°C. The solution is generally allowed
to
boil for about IO minutes. When the palladium and lipoic acid react, a clear
dark red solution is produced. The pH of the palladium-lipoic acid solution
18
WO 95/14466 PCT/US9411326U
2' 7 ~~~3
can be adjusted to at least about 6, preferably to a pH between about 6 and
about 7, and more preferably to a pH of about 6.8.
In the practice of the present invention, water may be added to the
palladium-lipoic acid solution in an amount sufficient to obtain a
concentration of the palladium-lipoic acid complex of at least about 0.01 M,
preferably between about 0.01 M and about 0.08 M, and most preferably in
an amount sufficient to obtain a concentration of about 0.04 M.
This resulting Pd-lipoic acid complex has been designated by the
present inventor as Polynucleotide Reductase Core, referred to herein as
"Poly-RC." Other ingredients may be added to Poly-RC to enhance its
chemical and biological activity. For administration to humans, the pH of
the complex is preferably adjusted to about 6.8 by the addition of an acid, if
necessary, and the concentration is preferably adjusted to about 0.04 M by
the addition of a sufficient amount of water. This solution of the complex is
stable and may be easily stored at room temperature.
One preferred embodiment of the present invention comprises a Pd-
lipoic acid complex to which thiamine or a salt thereof has been added. The
thiamine salt will be bound to the palladium metal ion at the Nl nitrogen of
the pyrimidine ring of thiamine. Hadjiliadis et al., "Interaction of Thiamine
and its Phosphate Esters with Pt(II) and Pd(II)," Inorganica Chimica Acta
25L21-3I (1977). Any thiamine salt is useful in the practice of the present
invention. Preferably, the thiamine salt is selected from thiamine
hydrochloride, thiamine nitrate, thiamine phosphate, and thiamine
19
WO 95/14466 ~ PCT/US94/13260
pyrophosphate. Most preferably, the thiamine salt is thiamine
hydrochloride. The thiamine or a salt thereof may be added to the Pd-lipoic
acid solution after the Pd-lipoic acid solution is cooled to at least about
55°C, preferably to between about 20° and about 50°C, and
most preferably
to about 42°C. The solution may be cooled by any method known in the
art.
While the thiamine or salt~thereof is being added to the solution
containing the palladium-Iipoic acid complex, mixing of the thiamine with
the Pd-lipoic acid solution can be facilitated, for example, by stirring or
agitation of the solution. The resulting solution may then be filtered,
preferably sterile-filtered, by methods known in the art. The resulting novel
composition has been designated by the present inventor as Polynucleotide
Reductase-2, referred to herein as "Poly-R2~~. The pH of Poly-R2 is
adjusted to at least about 6, preferably to a pH between about 6 and about 7,
and more preferably to a pH of about 6.5.
Sufficient water should be added to the palladium-lipoic acid solution
to obtain a concentration of the palladium-lipoic acid-thiamine complex in
the Poly-R2 of at least about 0.01 M, preferably between about 0.01 M and
about 0.08 M, and most preferably in an amount sufficient to obtain a
concentration of about 0.04 M in the resulting composition. Poly-R2 is
stable and may be easily stored at room temperature.
A further preferred embodiment comprises a thiamine salt of the Pd-
lipoic acid complex to which a synthetic, i.e., activated, cofactor of vitamin
WO 95/14466 PCT/US94/13260
2~~6~0~
B12 (cyanocobalamin) is added. The vitamin B12 cofactor of the complex is
cyanocobalamin which has been reacted with acetylcysteine. This thiamine
salt of the Pd-lipoic acid complex having an activated cofactor of vitamin
B12 has been shown to exhibit even more enhanced biological activity than
the Poly-RC and Poly R2. This Pd-lipoic acid derivative is referred to
herein as B12AC-Poly-R2.
The activated B12 is prepared by mixing equal amounts of
cyanocobalamin and acetylcysteine in a sufficient amount of water to
dissolve both of the substances. The pH of the activated B12 solution is
preferably adjusted to at least about 6, preferably to a pH between about 6
and about 7, and more preferably to a pH of about 6.5. For example,
NaOH may be added to the solution to adjust the pH to about 6.5 in a
particularly preferred embodiment. The solution is then boiled for about 10
minutes.
The activated B 12 is added to the Poly-R2 solution prior to the
addition of water to adjust the concentration of the Poly-R2. The amount of
activated B12 added is generally in the range of about 0.1 to about 5 grams
in 20 liters of Poly-R2. Sufficient water should then be added to the solution
to obtain a concentration of the activated B12-palladium-Iipoic acid-thiamine
complex in the B12AC-Poly-R2 of at least about 0.01 M, preferably between
about 0.01 M and about 0.08 M, and most preferably in an amount sufficient
to obtain a concentration of about 0.04 M in the resulting composition.
B12AC-Poly-R2 is stable and may be easily stored at room temperature.
21
WO 95/14466 2 i 7 b 6 0 3 pCT/US94/13260
The palladium-lipoic acid complexes, e.g., Poly-RC, Poly-R2 and
B12AC-Poly-R2, may be identified using W-visible spectroscopy, and
preferably by cyclic voltammetry, as discussed further in the Examples. The
structures of these complexes, as shown in the Examples, were also studied
by Fourier transform-infrared spectroscopy (FTIR). Cyclic voltammetry was
performed to demonstrate the charge interactions of the palladium-lipoic acid
complexes with DNA or RNA. Such studies were performed using Poly-
RC, Poly-R2 and B12AC-Poly-R2 with DNA and Poly-R2 with RNA. These
studies illustrated that the palladium-Iipoic acid complexes of the present
invention shunt electron energy from the complexes to nucleic acids of DNA
or RNA and are polynucleotide reductases. The results of these studies are
further discussed in the Examples.
The present inventor has also found that these novel polynucleotide
reductases induce new varieties of cell forms in amoeba, yeast and mold.
For example, these reductases produce giant Baker's yeast cells. They also
induce micronucleated nuclei in Baker's yeast, amoeba, and tumors. In
addition, the polynucleotide reductases of the present invention cause
formation of multiple spore bearing sori on the stalks of the mold
Dictyostelium discoideum.
These novel polynucleotide reductases have also been found to induce
dense granular chromatin in the nuclei of amoeba, yeast and mold. The
dense form of chromatin produced in cells is generally referred to as
heterochromatin. Manuelidis, "A View of Interphase Chromosomes"
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WO 95/14466 PCTIUS94/13260
fence 250:1533-1540 (December 14, 1990); and Yunis et al.,
"Heterochromatin, Satellite DNA, and Cell Function" ~rign_~g 174:1200-
1208 (December 17, 1971). During cell replication, for the chromosomes to
condense in metaphase, metabolic energy is required. As the condensation
of chromatin is lrnown to be energy dependent (Manuelidis et al.), these
studies would further support the inventor's belief that the effect of Poly-
RC,
Poly-R2 and B12AC-Poly-RZ on DNA and RNA is an electron transfer
reaction.
While not wishing to be bound by any theory, the present inventor
believes that the novel polynucleotide reductases induce energy dependent
conformational changes in DNA and RNA. These conformational changes in
DNA and RNA result from the nucleic acids being in a more reduced state
as a result of electron transfer upon interaction with the polynucleotide
reductases of the present invention.
The present invention also provides a pharmaceutical composition of
matter comprising a pharmaceutically effective amount of the complexes
previously described, e.g., the palladium or a salt thereof and lipoic acid or
a derivative thereof complex, the palladium-lipoic acid complex which
further comprises thiamine or a salt thereof, or the thiamine-palladium-lipoic
acid complex which further comprises a synthetic cofactor of vitamin B12
(cyanocobalamin), and a pharmaceutically acceptable carrier therefor.
Preferably, the pharmaceutical composition comprises a (palladium)m(lipoic
acid)n complex, wherein m and n are each independently 1 or 2, and more
23
WO 95/14466 PCT/US94/13260
preferably, wherein m is 1 and n is 1. In a further preferred embodiment,
the pharmaceutical composition comprises the palladium-lipoic acid complex
in a solution in an amount sufficient to obtain a concentration of about
0.04 M. For the pharmaceutical composition comprising the thiamine-
palladium-Iipoic acid complex which further comprises a synthetic cofactor
of vitamin B12 (cyanocobalamin), 1000~cg of vitamin B12 (cyanocobalamin)
will preferably be present in a 10 ml dose of the thiamine salt of the
palladium-lipoic acid complex.
The pharmaceutically acceptable carriers of the present invention
depend on the dosage form selected and are carriers which are well known
in the art. Different routes of administration necessarily require different
pharmaceutically acceptable carriers. An identification of such carriers may
be found in any standard pharmacy text, for example, Remineton's
Pharmaceutical Sciences, 17th edition, ed. Alfonso R. Gennaro, Mack
Publishing Company, Easton, PA (1985). More specifically, examples of
pharmaceutically acceptable carriers include pharmaceutical diluents,
excipients or carriers suitably selected for the intended route of
administration which is consistent with conventional pharmaceutical practice.
For instance, for oral administration in the form of tablets or capsules, the
active drug components may be combined with any oral non-toxic
pharmaceutically acceptable inert carrier such as starch, cellulose,
magnesium stearate, dicalcium phosphate, calcium sulfate, mannitol, and the
like. Moreover, when desired or necessary, suitable binders, lubricants,
24
CA 02176603 2004-03-04
WO 95/14466
PCTIUS94/13260
disintegrating agents and coloring agents can also be incorporated in the
mixture.
Suitable binders, for example, include starch, gelatin, natural and synthetic
gums
such as acacia, sodium alginate, carboxymethylcellulose, polyethylene glycol
and
waxes. Among the lubricants, there may be mentioned for use in these dosage
forms, boric acid, sodium benzoate, sodium acetate, sodium chloride, etc.
Disintegrators include, without limitation, starch, methylcellulose, agar,
bentonite,
guar gum, etc. Flavoring agents and preservatives can also be included where
appropriate. In the case of tablets, they can be further coated with the usual
coating
materials to make, for example, sugar-coated tablets, gelatin film-coated
tablets,
tablets coated with enteric coatings, tablets coated with films or double-
layered and
multi-layer tablets.
For parenteral administration, for example, the formulations must be sterile
and pyrogen-free, and are prepared in accordance with accepted pharmaceutical
procedures, for example, as described in Reminc~ton's Pharmaceutical Sciences
17tn
Edition, Alfonso R. Gennaro, Mack Publishing Company, Easton, Pa (1985) at pp.
1518-1522. The aqueous sterile injection solutions may further contain anti-
oxidants, buffers, bacteriostats, isotonicity adjusters and like additions
acceptable for
parenteral formulations. Various unit dose and multidose containers, e.g.,
sealed
ampules and vials, may be used, as is well-known in the art. The essential
ingredients of the sterile parenteral formulation, e.g., the water and the
selected
palladium-lipoic acid complex, may be presented in a variety of ways, just so
long as
the solution ultimately administered to the patient contains the appropriate
amounts
of the
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WO 95/14466
PCT/US94/13260
essential ingredients. Thus, for example, the palladium-lipoic acid
complex/water formulation may be presented in a unit dose or multidose
container, ready for injection. As another example, a concentrated solution
of palladium-lipoic acid complex/water may be presented in a separate
container from a diluting liquid (water or palladium-lipoic acid
complex/water) designed so that the contents can be combined to give a
formulation containing appropriate amounts for injection. As another
alternative, the palladium-lipoic acid complex may be provided in a freeze-
dried condition in one container, while a separate container contains diluting
liquid (water~or palladium-lipoic acid complex/water, depending on the
amount of palladium-Iipoic acid complex in the other container), again
designed so that the contents can be combined to give a formulation
containing the appropriate amounts of the water and selected palladium-Iipoic
acid complex. In any event, the contents of each container will be sterile.
Suitable carriers for parenteral administration include, for example, water,
ethyl alcohol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated
isostearyl alcohol, polyoxyethylene sorbitol and sorbitate esters. In these
instances, adequate amounts of sodium chloride, glucose or glycerin can be
added to make the preparations isotonic.
As previously stated, the synthetic polynucleotide reductases have
been found to be useful as therapeutic agents for cancer and the treatment of
psoriasis. Data reporting therapeutic effects are best presented by
independent institutional protocols. Compassionate Investigational New
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WO 95/14466 217 b 6 fl 3 PCT/US94/13260
Drug studies in humans involving administration of the novel polynucleotide
reductases were begun in November of 1992 and are presently ongoing. As
of February 3, 1993, eighty patients with tumors have been treated with
Poly-R2. As of the filing date of the present application, approximately 800
patients have been treated with B12AC-Poly-R2. These terminally ill patients
have experienced a reduction in tumor size by objective criteria and
subjective relief of symptoms caused by various cancers.
For example, Poly-R2 has also been administered to patients having
metastatic lesions to the liver. In these patients, the serum albumen levels
return to normal after treatment with Poly-R2. Moreover, the enlargements
of the liver become no longer palpable.
With respect to the range of tumor types responding to Poly-RC,
Poly-R2, and B12AC-Poly-R2, oncologists are using these polynucleotide
reductases "across the board," e.g., for treatment of carcinomas and
adenocarcinomas of the lung, breast, colon, esophagus, or pancreas;
malignant melanomas; liver metastases; or AIDS-related lymphomas or
sarcomas. Specific lesions undergoing liquefaction at this time include
adenocarcinomas of the breast and bowel, and pancreatic and lung
carcinomas. Malignant melanomas and liver metastases are also presently
being treated with Poly-RC, Poly-R2, and B12AC-Poly-R2. Such
carcinomas, adenocarcinomas, melanomas and metastases are shrinking and
developing non-hemorrhagic central liquefaction after treatment with
Poly-RC, Poly-R2, and B12AC-Poly-R2. Bi2AC-Poly-R2 is particularly
27
WO 95/14466 217 6 6 0 3 pCT/US94/13260
preferred for the above methods of treatment. Oncologists have also stated
that the pattern of their practice has changed as there are now fewer
weekend emergencies involving the oncological patients being treated with
these polynucleotide reductases.
Bi2AC-Poly-R2 has also been found useful for the treatment of
psoriasis. For the treatment of psoriasis, the B12AC-Poly-R2 is preferably
topically administered to the afflicted area of the patient, however, other
forms of administration including oral and parenteral may be useful in the
practice of the present invention. Examples of types of psoriasis which may
be treated include psoriasis wlgaris. Psoriasis wlgaris has been treated
topically with a 0.04 M B12AC-Poly-R2 solution. At present, 10 patients
have received B12AC-Poly-R2 for the treatment of psoriasis. Patients with
severe psoriasis having thick, large plaques involving greater than 40 % of
their body surface were chosen for treatment. After one week of treatment
involving application of B12AC-Poly-R2 one to two times daily, significant
objective improvement of psoriatic lesions were observed. Scaling,
thickness, roughness and inflammation of psoriatic plaques were reduced by
at least SO % . In some treated areas, the psoriatic plaques have been
sufficiently reduced for normal skin markings, e.g., stretch marks and skin
creases, to again be evident in the lesional areas. Figures 10A, lOB, IOC
and lOD illustrate the effect of B12AC-Poly-R2 when used to treat severe
psoriasis. While no treated plaques have been completely resolved and
cleared, the positive therapeutic effects observed after a relatively short
28
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WO 95/14466 PCTIUS94113260
period of topical treatment have been quite significant. Further prolonged
studies of the effects of Poly-R2 on psoriasis are still ongoing as of the
filing
date of this patent application.
The dosage of the compositions of the present invention is selected,
for example, according to the usage, purpose and conditions of symptoms.
Furthermore, the dose administered will be selected, for example, according
to the particular composition empl6yed and the size and condition of the
patient as well as the route of administration employed, but in any event will
be a quantity sufficient to cause a reduction in tumor size or a reduction of
maculopapules associated with psoriasis.
For tumors, Poly-RC, Poly-R2 or B12AC-Poly-R2 is administered to
a patient in an amount effective for tumor reduction to a patient in need of
such treatment. A parenteral route of administration is preferred including,
for example, intravenous, intramuscular, subcutaneous, intradermal, topical,
intrathecal and intraarterial methods. More preferably, the pharmaceutical
composition of the present invention is parenterally administered to a patient
at a dosage of between about 5 and about 30 ml daily of a 0.04 M solution
of the pharmaceutical composition for at least about 5 days. At present, the
prevailing dosage pattern in adult humans has been 40.0 ml of 0.04 M Poly-
RC, Poly-R2 or B12AC-Poly-RZ administered daily for the first three days of
treatment, followed by 20.0 ml daily for an additional 14 days of treatment.
Alternatively, a pharmaceutical composition comprising B12AC-Poly-R2 may
be administered to a patient at a dosage of about 40 ml for about 10 days.
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WO 95!14466 PCT/US94/13260
However, the precise route of administration, dosage and frequency of
administration is individualized for each patient and can vary over a wide
range depending on the particular disease state being treated, the condition
of
the patient and the like.
For the treatment of psoriasis, the pharmaceutical compositions of the
present invention are administered in an amount effective for reduction of
maculopapules associated with psoriasis to a patient in need of such
treatment. Parenteral routes of administration, in particular topical, are
preferred. Preferably, the thiamine salt of the palladium-Iipoic acid
complex, and~ more preferably the thiamine salt of the palladium-lipoic acid
complex which further comprises a synthetic cofactor of vitamin B12, is
topically administered to a patient at a dosage of between about 5 and about
80 ml daily of a 0.04 M solution for at least about 5 days. For psoriasis,
the prevailing dosage has been between about 5 and about 10 ml of
B12AC-Poly-R2 topically administered one to two times daily on the area to
be treated for about one week. A 10 ml dose of B12AC-Poly-R2, for
example, will comprise approximately 210.0 mg of the thiamine salt of a
palladium-lipoic acid complex composition and 1000 ~cg of the activated B12
compound. Subsequent dosages are then determined by observation of the
response to the composition and further evaluation of the patient. Once
again, treatment is individualized. The patient may be reevaluated at any
time in order to determine whether treatment should be continued and at
what dosage.
WO 95/14466 PCTILTS94/13260
Higher dosages of the palladium-lipoic acid complexes, or
polynucleotide reductases, are generally administered intravenously, while
lower dosages may be given by any injectable route. There has been no
observable toxicity at the dosages presently contemplated, including 100.0 ml
of 0.04 M solution of B12AC-Poly-R2 administered intravenously. The
intraperitoneal LD 50 in 20.0 g hybrid Swiss mice is 0.80 ml of this
solution, which extrapolates to approximately 800.0 mI in humans. The
dosage range of the novel palladium-lipoic acid complexes, or polynucleotide
reductases, of the present invention is about 500 to about 2000 mg daily.
Treatment including dosages and routes of administration as indicated above
is individualized and is determined by a clinician and depends on a variety of
factors including the specific disease state treated, the condition of the
patient, and the like.
The present invention also includes pharamceutical compositions
comprising the novel palladium-lipoic acid complexes as previously
described supra. In general, the pharmaceutical compositions of the present
invention may be administered by any parenteral route, with intravenous,
intramuscular, subcutaneous, intradermal, topical, intrathecal and
intraarterial methods being preferred for the treatment of tumors. At
present, for example, up to 100.0 ml of B12AC-Poly-R2 has been injected
intravenously without any adverse response by the treated patient. Oral
forms of palladium-lipoic acid complexes may also be suitable for the
practice of the present invention. For the treatment of psoriasis, topical
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WO 95/14466 PCT/US94/13260
administration of the pharmaceutical compositions of the present invention to
the affected area is preferred.
In order to further illustrate the present invention, the following
specific examples are given, it being understood that the same are intended
as illustrative and in nowise limitative.
~XAMPLE~
x m 1 1
Prevaration of Polynucle~t~dp 1?edu~tase fPolv R~~
The following substances were obtained for laboratory use:
de-ionized distilled water, palladium dichloride (Sigma) and lipoic acid
(Fluka).
A solution of 80.0 ml of 1.0 N HCl was placed in a 2000 ml
multi-necked spherical glass reactor vessel in a hemispheric heater. 7.10 g
of PdCl2 was then added to the HCl solution. The reactor vessel was stirred
with a lightning type mixer. The shaft and rotor of all reactors were
plastic-coated so that no metal was exposed to the solutions. The solution
was brought to a gentle boil for ten minutes. The boiling temperature was
close to that of water, e.g., about 100°C. The color of the solution
upon
boiling was a clear dark amber. Once a clear, dark amber solution was
achieved, this solution was removed from the reactor, filtered to clarity, and
replaced in the rinsed 2000 ml reactor.
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WO 95/14466 / ~ PCT/US94113260
In a separate beaker 8.26 g of lipoic acid was stirred and dissolved in
285 ml of 1.0 N NaOH. If any undissolved residue remained, the solution
was filtered to complete clarity. ,
The dissolved lipoic acid was next added to the palladium solution
with stirring and continued heating until the solution was brought to a gentle
boil. The boiling temperature is close to that of water, e.g., 100°C.
After
ten minutes of boiling, a clear dark red solution was produced. This
solution contained the essential core complex: Pd-Iipoic acid, i.e., Poly-RC.
The pH of the Poly-RC was adjusted to 6.8 by the addition of 1.0 N
HCI. Water was added to adjust the volume to 1000 ml. The concentration
of the resulting palladium-lipoic acid complex was 0.04 M.
Example 2
~reoa_ration of Pol~nucleotide Reductase-2 fPoly-R21
In order to prepare Polynucleotide Reductase-2 (Poly-R2), 9.0 g of
thiamine-HCI (Fluke), was dissolved in 200 ml water.
When the red Pd-lipoic acid solution containing the essential Pd-lipoic
acid core complex produced in Example 1 had cooled to 42°C, the
thiamine-
HCl solution was added with vigorous stirring. The pH was 6.5. The total
volume of the thiamine salt of a palladium-lipoic acid complex solution was
adjusted to 1000 ml by the addition of water. The solution was next
sterile-filtered through a 0.2 micron pore membrane flask. Water was added
to adjust the volume to 1000 ml, resulting in a 0.04 M solution of Poly-R2.
33
WO 95/14466 PCT/US94113260
x m le
Preparation of B12AC-polv-R2
Tn order to prepare B12AC-Poly-R2, 100 mg of cyanocobalamin
(Sigma), was mixed with 100 mg of acetylcysteine (Fluka), and dissolved in
20.0 ml of water. The pH was adjusted to 6.5 by the addition of NaOH,
and the resulting solution was then boiled for 10 minutes. The solution was
cooled and then mixed with the Poly-R2 which was obtained prior to
adjusting the volume to 1000 ml in Example 2. Water was next added to
adjust the volume to 1000 ml, resulting in a 0.04 M solution of B12AC-
Poly-R2 based on the Poly-R2 concentration. The cyanocobalamin
concentration was 0.1 mg/ml.
xam le 4
Identification of Potv R~ pm,~ n~ ., a
~~. mu r~12~-pOlV-RZ
L~sina W-vi~;hle cpectrOSCnnv
An ultraviolet spectra of uncomplexed sodium lipoate in
solution (10~ ~c) was first conducted using a Shimadzu UV-160U recording
Spectrophotometer. This spectra is shown in Figure 2A. This figure shows
that UV absorbance peaks do not result from a solution of uncomplexed
sodium lipoate. In addition, the almost clear solution had no absorbance
peaks in the visible region.
The ultraviolet spectra of Poly-RC was observed on an Hitachi
34
217~~Q3
WO 95/14466 PCT/US94/13260
model 440 Spectrometer with a 2 nm bandpass and is shown in Figures 2B
and 2C. For the spectra of Figure 2B, a 2x10' ~c solution was used, while
for Figure 2C, a 10'~ ~c solution was used. The resulting peaks may be
interpreted using any spectrophotometry text, for example Shugar et al., ~
Chemist's ReadX,Reference Handbook, McGraw-I3i11, p. 6.19 (1990).
Poly-RC had absorbance peaks at 200, 205, and 261 nm and minima
at 197, 202, and 253 nm.
After ninety days of air oxidation of the Poly-RC solution, an
ultraviolet spectra was again obtained on the Shimadzu UV
spectrophotometer. These spectra are shown in Figures 2D and 2E. In both
instances, a 1.2x10' ~c solution was employed. As can be seen in Figure
2D, Poly RC is in the oxidative state which is evidenced by the UV
absorbance peaks at 240 nm and 289 nm. Figure 2E illustrates the visible
range of the Poly RC, in which range an inflection is estimated as occurring
at 396 nm. These spectra showing a shift from the fresh Poly RC solution
are consistent with the view that Poly RC is a redox complex.
Stoichiometric studies of various Poly-RC solutions were performed
in the visible range. Figure 2F shows the visible absorbance spectra of the
palladium-Iipoic acid complex made at various ratios of Iigand to metal.
This preliminary work suggested that there are at least two forms of
Pd-lipoic acid complex (as previously illustrated in the specification): (1)
A 1:I ratio of palladium to Iipoic acid (nl), and (2) A 1:2 ratio of palladium
to lipoic acid (n2). The 1:2 form showed a stronger red-shifted absorbance.
SUBSTITUTE SHEET (RULE 26)
WO 95/14466
2 ~ 7 6 ~ 0 3 PCT/US94/13260
Interestingly, however, this 1:2 form appeared to have less biological
activity. This suggested that the second lipoic acid competes with the
biologic site for interaction with the palladium. The 1:3 mixture of Pd-lipoic
acid (n3) showed an unexpected intermediate absorbance curve. While this
requires further study, it is presently viewed to be a mixture of the
palladium-lipoic acid complexes having 1:1 and 1:2 ratios of Pd to lipoic
acid.
Poly-R2 differs from Poly-RC by the addition of thiamine-HCl to the
Pd-lipoic acid solution. It is notable that the palladium chloride solution
forms a bright red solution when it is mixed with the thiamine-HCl solution
with a Pd:thiamine ratio of 1:2, and the pH is adjusted to 6.5 with NaOH.
Figure 2G shows the absorbance spectra of a 2x10' ~c solution of Poly-R2.
The spectral character indicates that the thiamine interacts with palladium
and can form interactive salts such as the thiamine salt of a palladium-Iipoic
. acid complex.
As can be seen in Figure 2H, the peak absorbance of bis(thiamine
HCl)Pd is 355 nm. For this spectra, a SxIO'~ ~c solution was used. Figure
2I compares the spectra of vitamin B12 and the activated cofactor thereof
which is included in the BI2AC-Poly-R2 of the present invention.
For the Pd-lipoic complex of the present invention, the molecular
weight for a 1:1 ratio was calculated to be 312.73, although the possibility
of
polymers of this structure exists, e.g., X(1:1). At a wavelength of 261 nm
36
WO 95/14466 217 6 6 Q 3 PCT/US94/13260
the absorptivity of the complex (a=A/bc) was 1.1 x 105. The molar
absorptivity (e=a x mol. wt.) was calculated to be 3.44 x 10~.
x m 1
Structure of the Palladium-Lipoic Acid Comb
The structure of the palladium-lipoic acid complex was studied by
Fourier transform-infra red spectroscopy (FT'IR). To perform this study, the
red solution containi;~ ~ the essential core complex of palladium-Iipoic acid,
i.e., Poly-RC, was evaporated to a stable weight residue by heating the
solution in a vacuum. The residue was first mixed with KBr and
compressed to a table: and was then scanned in a Perkin-Elmer FTIR
spectrophotometer.
Uncomplexed Iipoic acid was similarly treated. The two FTIR scans
are compared in Figures 3A, 3B and 3C. Characteristic frequencies of the
new complex are marked with arrows. Contributing frequencies of lipoic
acid are marked with triangles in the figures.
There were interactions showing changes at six frequencies. The new
peaks (ciri l) were interpreted according to Nakamoto, Infrared and Raman
Sgectra of Inoganic and Coordination Compounds, 4th, John Wiley & Sons
(1986); Silverstein et al., spectrometric Identification of Organic
Compounds, 3d, John Wiley & Sons (1974); Flett, Physical Aids to the
Organic Chemist, Elsevier, NY, 162-163 (1962); Schotte, L.,
"Spectrochemical Studies on Disulphides, 1. An Investigation of Some
37
WO 95/14466 217 6 6 Q 3 PCT/US94/13260
Linear and Cyclic Carboxylic Substituted Disulphides with Special Reference
to the 1,2 Dithiolane System," Ark. Kemi, Vol. 8, No. 56, p. 579-596,
(1955); and Schotte, L., "Studies on Sulphur Compounds Related to Glutaric
and Pimelic Acid with Special Reference to the 1,2-Dithiolane System," ~,rk,
emi, VoI. 9, No. 37, pp. 441-469 (1956), and were as follows:
645 - sulfide stretch, presumably to the metal
865 - -CH2 deformation, consistent with chain bend
1040 - S-0 stretch,
1420 - C-0 stretch, presumably oxygen to metal, also CH2 bend
1440 - C-0 stretch, presumably oxygen to metal, also CH2 bend
1575 - C-0 stretch, presumably oxygen to metal
The contributing IR frequencies from the spectra of free lipoic acid
are as follows:
1700 - COO', and C=O
1430 and 1410 = CH2 scissor or bend, also C-O symmetric stretch
930 - C-C stretch
730 and 680 - C-S stretch
480 - S-S stretch
1350 and 1250 - CHZ twist
A depiction of this structure is shown, together with the variant
having two Iipoic acid moieties, on page 9.
According to the infra-red spectra, the complex between palladium
and lipoic acid is bonded: (1) at the carbonyl of the carboxyl group with
3'8
WO 95/14466 217 b ~ 0 .~ PCT/US94/13260
probable resonance involvement of both oxygens, and (2) at one or more
sulfur atoms. In addition, there was a steric assistance by Iipoic molecular
bending favored by the sulfur to oxygen stretch. Bending was also indicated
by the -CH2 deformation.
The result is a bent chain of Iipoic acid, with its ends bonded by way
of palladium coordination. This 1:1 structure is represented as a bent cyclic
structure as previously illustrated. A 1:2 palladium to lipoic acid complex is
also possible as illustrated. Crystallographic studies show that the palladium-
lipoic acid complexes form trigonal prisms as shown in Figure 1. Since
redox reactions involving the Pd-lipoic acid complex occur, the structure
may also be represented by its oxidized and reduced forms, which increases
the number of possible structures of the complex.
x m le
Identification of the Complexes
Voltammetry was also used to identify the complexes of the present
invention. Cyclic voltammetry of the reductases was performed on an
EG&G model 264A polarographic system as follows:
The working electrode used a static mercury drop, the reference
electrode was Ag/AgCI wire in KCl solution housed behind a porous Vycor
frit and the counter electrode was Ag/AgCI wire. A reversing scan was
performed from either 0 or -0.3 Volt to -1.0 Volts at 20 uA or 100 uA
sensitivity and a scan rate of 100 mv/sec. Before the reductase sample was
39
WO 95/14466 ~ ~ PCT/US94/13260
run, 9.0 ml of acetate buffer background electrolyte at pH 5.0 was purged
for 8 minutes by vigorous bubbling of nitrogen. The addition of each
sample was followed by two minutes of nitrogen purging. Figures 4A, 4B
and 4C show the resulting cyclic voltammetric scans of Poly-RC and
Poly-RZ. Figure 4A shows the resulting cyclic voltammetric scan of Poly-
RC (.12 ~c) from 0 to -1.0 volts at 20 ~.A sensitivity. Figure 4B shows the
scan of Poly-R2 (.08 ~,) from 0 to -1.0 volts at 100 ~,A sensitivity. Figure
4C shows the scan of Poly-R2 (.08 ~c) from -.3 to -1.0 volts at 20 ~cA
sensitivity.
The scans were quasi-reversible with proportionally undersized anodic
peaks. Cathodic peaks with a common base typifying catalytic activity were
characteristic of the reductases. Figure 4A shows peaks in poly-RC at -660
and -800 mv. In Figure 4B, with slightly different parameters, Poly-RZ had
an enormous catalytic triple peak at -160, -200, and -218 mv. This was due
to the addition of thiamine-HCl and was recorded at 100 uA sensitivity. The
remaining two peaks were at -718 and -800 my and are shown in Figure 4B,
and then more graphically in Figure 4C at 20 uA sensitivity. Figures 4E
and 4F illustrate the electrochemical signatures for B12AC-Poly-R2 (.04 ~c).
The peaks for this complex were at -150, -200, -220, -705 and -845 my and
are shown in Figure 4E from -.3 to -1.0 volts at 100 uA sensitivity, and then
more graphically in Figure 4F from 0 to -1.0 volts at 20 uA sensitivity.
These scans in Figures 4A, 4B, 4C, 4E and 4F thus represent the
identifying electrochemical signatures of these novel compounds of the
WO 95/14466 ~ PCT/US94/13260
present invention. In addition to characteristic reactions of these reductases
with nucleic acids, the electrochemical signatures allow identification of the
novel polynucleotide reductases of the present invention.
With respect to the interaction between palladium and thiamine, these
materials act as a redox pair which shifts the peaks of Poly-RC as measured
by voltammetry to form the new Poly-R2 peak at -718 my as is shown in
Figure 4D.
xam le 7
charge Interactions of DNA Reductases
To demonstrate the charge interactions of DNA reductases, cyclic
voltammetry was performed on the interactions of the Pd-lipoic acid
reductases with DNA or RNA. Figure 5 shows the charge interaction of
Poly-RC with DNA. DNA has no electro-chemical signal of its own in this
range. In this example, 1.0 ml of a 5 mglml DNA solution was added to
250 ~cl of a .12 ~c Poly-RC solution. In this voltammetric study the Poly-RC
baseline (1), the transient effect of the addition of DNA on the baseline (2),
and the equilibrium effect of the addition of DNA (3) were evident. The
double peaked catalytic wave of Poly-RC was altered on the addition of
DNA. This scan was run from 0 to -I.0 volts at 20 ~cA sensitivity.
The more reduced (-) peak became diminished and the more oxidized
(+) peak became transiently increased, and was further decreased when
41
WO 95/14466 PCT/US94/13260
equilibrium was reached. Thus current had dropped and voltage had moved
in the oxidative direction.
These voltammetry studies, therefore, showed that DNA had acted as
an oxidant. The amount of energy transferred is calculated from the shift in
the voltage and current coordinates in terms of the Nernst relation. This
interpretation of shifts in electro-potential curves follows the
interpretations
of electrochemistry literature, e.g., Maloy, "Factors Affecting the Shape of
Current Potential Curves," J. Chem. Ed , 60(4):285-289 (April 1983);
Rieger, Electrochemi trv, Prentice Hall (1987); and Riley et al., rin i le
9f Ele~tro alvtical_ Methods, John Wiley (1987).
Figures 6A and 6B show a similar pattern for Poiy-R2 interaction
with DNA. Figure 6A was run from 0 to -1.0 volts at 100 ~cA sensitivity,
while Rigure 6B was run from -.3 to -1.0 volts at 20 ~cA sensitivity. In both
figures the Poly-R2 baseline (1), the transient effect of the DNA (2), and the
equilibrium effect of the DNA (3) were evident. 1.0 ml of a 5 mg/ml DNA
solution was added to .3 ml of a .08 ~c Poly-R2 solution. DNA again acted
to suppress current peaks and shift the voltage in the (+) direction. For
Poly-R2 the shifts were of greater magnitude. Figure 6C shows another
similar pattern of reaction for Poly-R2 (.3 ml of a .08 ~ solution) and yeast
RNA (1.0 ml of a 5 mg/ml solution) with a scan from -.3 to -1.0 volts at 20
~cA sensitivity. In this figure (1) represents the Poly-R2 peak. These figures
thus evidence that Poly-R2 can reduce both DNA and RNA.
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WO 95/14466 217 b 6 0 3 PCT/LTS94/13260
Figure 7A shows cyclic voltammetry of the interactions of Poly-R2
(.3 ml of a .08 ~c solution), DNA (1.0 ml of a 5 mg/ml solution) and vitamin
B12 (cyanocobalamin) (1.0 ml of a .2 mglml solution). This can be
compared to Figure 7B which shows the interactions of B12AC-Poly-R2 (.5
ml of a .04 ~c solution) with DNA (.5 ml of a 5 mglml solution). Both scans
were run from -.3 to -1.0 volts at 20 ~cA sensitivity. As can be seen by the
two figures, the interaction of Poly-R2 with the activated form of B12 and
DNA will reach equilibrium much more rapidly than Poly-R2 with vitamin
B12. In Figure 7A, the baseline signal marked (1) in the figure was oxidized
by DNA to the first equilibrium at (2). At this point, addition of vitamin
B12 Produced a further oxidation which reached equilibrium at (3). In
Figure 7B, the baseline signal marked (1) in the figure was oxidized by
DNA to equilibrium at (2). Thus, equilibrium was rapidly achieved. This
effect of B12 was restricted to DNA, and was not demonstrable in the RNA
, oxidation of Poly-R2. These data have implications regarding the
biochemistry of vitamin B12 and suggest its possible role as a hydrogen
receptor for DNA. For example, vitamin B12 has recently been reported as
a radical trap by Finke et al. , "Radical Cage Effects in Coenzyme B 12~
Radical Trapping, Product and Kinetic Studies," J. Inorg~. Biochem., 51(1
and 2):221 (August 1993).
43
WO 95114466 21 ~ 6 6 (~ 3 pCT/US94/13260
x m 1 8
Induction of New Yeast Form
Studies conducted by the present inventor illustrated that
polynucleotide reductases induce new varieties of cell forms in amoeba,
yeast, and mold. Brewer's yeast (Saccharomyces cerevisae) was cultured in
2.0 % malt extract and 2.0 % sucrose. Poly-R2 was added to achieve a
concentration of 10'~ M.
The yeast was incubated for 30 minutes at 28°C and examined under
phase microscopy. Figure 8A illustrates the altered cell morphology of the
yeast cells. ~ Large flat cells were one kind of new variety produced by the
addition of Poly-R2. Figure 8B shows a stage in the giantization of these
cells in which Iipid droplets are lost from the cell. The demulsification of
lipid may be a result of the loss of membrane charge. This cell variety is
not described in the group of letters to Science on the history of yeast
morphology found in Witkus, Steensma et al., Berbee et al., cien e,
257:1610 (Sept. 18, 1992).
Figure 9 shows the effect of vitamin B12 and Poly-R2 on Baker's
yeast. Exaggerated heterochromatin was produced by the addition of vitamin
B12 and Poly-R2 to Baker's yeast. In cells that have started to enlarge, there
was a dense condensation of chromatin at the nuclear membrane and
complete vacuolation of the remaining nuclear region. As can be seen in the
figure, some small cells had not yet developed these features.
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WO 95/14466 PCT/US94/13260
276603
xam le 9
Gomvassionate Investi~ational New Dru;~Study in Humans
Ten individuals having severe psoriasis with thick, large plaques
involving greater than 40% of their body surface currently undergoing
standard therapy, such as topical steroid, topical tar preparations, ultra
violet
light treatment, or methotrexate therapy, were chosen for an informal study
to investigate the benefits of B12AC-Poly-R2 in the treatment of psoriasis.
A particular area, most often the most recalcitrant and thickest
accessible plaque on the patient's body, was chosen for treatment. Each
patient was given S to 10 ml of B12AC-Poly-R2 which was applied with a
cotton-tipped applicator one to two times daily on the selected area for one
week. After the first application, a dramatic decrease in pruritus of the
lesion resulted almost immediately. Itching persisted, however, in the
untreated lesions. The area of application was also observed by the patients
to feel less tight, "less thick" and more supple.
Although one patient reported tightness, dryness and "crustiness" of
the treated area a few days after regular twice daily application of the
B12AC-Poly-R2, these side-effects disappeared when a moisturizer was used
by the patient. A reddish-brown stain resulted upon application of the
compound of the present invention, which washed off after 2 to 3 days.
Upon continuous topical administration one to two times daily of
B~2AC-Poly-R2 for one week, significant objective improvement of psoriatic
lesions were observed. Scaling, thickness, roughness and inflammation of
WO 95/14466 217 6 ~ D 3 PCT/US94/13260
psoriatic plaques were reduced by at least 50 °.'o . In some treated
areas, the
psoriatic plaques were thinned down to a sufficient degree that normal skin
markings, e.g., stretch marks and skin creases were again evident in the
lesional areas. While no treated plaques have been completely resolved and
cleared, these positive therapeutic effects observed after only one week of
topical treatment were significant. As of the filing date of this application,
six of the ten patients are presently undergoing further treatment with the
B 12~~-Poly-R2 composition of the present invention.
Figures 10A, lOB, lOC, 11A, 11B, 12A, 12B, I2C, 13A and 13B
illustrate photographs of patients oho have received topical
-:
treatment of BI2AC-Poly-R2. As can be seen in these figures,
after treatment with BIZAC-Poly-R2 the treated areas appear
smoother and less scaly as compared with the untreated lesions.
In Figure loA , an anterolateral abdomen afflicted with psoriasis
which has been treated with B12AC-Poly-R2 is shown on the right. This can
be compared with the left side of the photograph which shows an untreated
area. The area in loA was treated once daily for 7 days with BI2AC-Poly-
R2. By comparing the two sides of the photograph, it can be scen that the
treated psoriatic lesions appear less scaly, less thick and smoother. Stretch
marks of the surrounding and underlying skin are also more visible through
the thinner plaques of the treated region. In loB a close-up of an untreated
psoriatic plaque is set forth, showing thick, silvery scales with underlying
red inflamed base. In loc a treated area of the abdomen is shown. After 7
days of once daily treatment, less scaling and smoothening of lesions was
46
RECTlFlED SHEET (RULE 91)
WU 95/14466 ~ ~ PCT/US94/13260
evident. Again, normal skin markings are visible through the thinner
plaques. The redness is a stain from the application of the B12AC-Poly-R2.
Figure 11 compares an untreated llAand a treated 11B left lateral
thigh afflicted with psoriasis. The skin was treated with B12AC-Poly-R2
twice daily for 7 days. As can be seen in the figure, the treated area
appeared smoother and the plaques were thinner. The untreated area had
thick papulosquamous plaques showing silvery scales and a red inflamed
base.
Figure i2a illustrates a left lateral thigh with psoriatic plaque prior
IO to treatment. Figure 12B illustrates the same lesion after twice daily
treatment with B12AC-Poly-R2 for a 2 week time period. As can be seen by
comparison of the figures, the treated lesions appeared flatter and contracted
centrally with an overall diminution in size. In addition, overlying yellowish
"scabs" were evident, giving an impression of dryness. Superficial fissuring
may signify the drying effect and/or contraction of sections of the lesion.
One smaller lesion in the upper right quadrant remained unchanged. Figure
12c shows the same area as in its at a higher magnification. The abovc-
described features are even more evident at the higher magnification. In
addition, the small lesion in the upper left quadrant was superficially eroded
and appeared necrotic.
Figure 13A illustrates a dorsolateral view of forearms encased in
thick plaques of psoriasis prior to treatment with B12AC-Poly-R2. Figure
13s shows the same forearms one week later after treatment twice daily
47
RECTIFIED CHE1T (RULE 91)
WO 95114466 217 6 ~ D 3 PCT/US94/13260
with B12AC-Poly-R2 of the left forearm. The right forearm was untreated.
By comparison of the two forearms, it can be seen that the treated areas on
. the left forearm showed less scaling with smoothening and thinning of the
les~ns. ., In particular, improvement was noted on the area below the left
elbow.
Although the present invention has been described in connection with
preferred embodiments thereof, it will be appreciated by those skilled in the
art that additions, deletions, modifications, and substitutions not
specifically
described may be made without department from the spirit and scope of the
invention as defined in the appended claims.
48
