U.S. patent application number 10/824501 was filed with the patent office on 2004-09-30 for acyl deoxyribonucleoside derivatives and uses thereof.
This patent application is currently assigned to Wellstat Therapeutics Corporation. Invention is credited to Bamat, Michael K., Von Borstel, Reid W..
Application Number | 20040192635 10/824501 |
Document ID | / |
Family ID | 32330006 |
Filed Date | 2004-09-30 |
United States Patent
Application |
20040192635 |
Kind Code |
A1 |
Von Borstel, Reid W. ; et
al. |
September 30, 2004 |
Acyl deoxyribonucleoside derivatives and uses thereof
Abstract
The invention relates to compositions comprising acyl
derivatives of 2'-deoxyribonucleosides. The invention also relates
to methods of treating or preventing radiation, mutagen and
sunlight-induced cellular damage, methods for improving wound
healing and tissue repair, and methods for ameliorating the effects
of aging comprising administering the compositions of the present
invention to an animal.
Inventors: |
Von Borstel, Reid W.;
(Potomac, MD) ; Bamat, Michael K.; (Potomac,
MD) |
Correspondence
Address: |
NIXON & VANDERHYE, PC
1100 N GLEBE ROAD
8TH FLOOR
ARLINGTON
VA
22201-4714
US
|
Assignee: |
Wellstat Therapeutics
Corporation
Gaithersburg
MD
|
Family ID: |
32330006 |
Appl. No.: |
10/824501 |
Filed: |
April 15, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10824501 |
Apr 15, 2004 |
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09494242 |
Jan 31, 2000 |
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6743782 |
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09494242 |
Jan 31, 2000 |
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08153163 |
Nov 17, 1993 |
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6020320 |
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08153163 |
Nov 17, 1993 |
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07958598 |
Oct 7, 1992 |
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07958598 |
Oct 7, 1992 |
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07533933 |
Jun 5, 1990 |
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07533933 |
Jun 5, 1990 |
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07115923 |
Oct 28, 1987 |
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Current U.S.
Class: |
514/46 ;
536/27.3 |
Current CPC
Class: |
C07H 19/06 20130101;
A61K 45/06 20130101; C07H 19/10 20130101 |
Class at
Publication: |
514/046 ;
536/027.3 |
International
Class: |
C07H 019/16; A61K
031/7076 |
Claims
1-55 (cancelled).
56. A method for treating or preventing radiation-induced cellular
damage or sunlight-induced cellular damage comprising administering
to an animal an effective amount of a composition comprising an
acyl derivative of 2'-deoxyadenosine, having the formula 12wherein
R.sub.1, R.sub.2, and R.sub.3 are the same or different and each is
hydrogen or an acyl group derived from (a) an unbranched fatty acid
with 3 to 22 carbon atoms, (b) an amino acid selected from the
group consisting of glycine, the L forms of alanine, valine,
leucine, isoleucine, tyrosine, proline, hydroxyproline, serine,
threonine, cysteine, aspartic acid, glutamic acid, arginine,
lysine, histidine, carnitine, and ornithine, (c) nicotinic acid, or
(d) a dicarboxylic acid having 3 to 22 carbon atoms, provided that
not all of R.sub.1, R.sub.2, and R.sub.3 are H, and where R.sub.3
is not H, then R.sub.1 and/or R.sub.2 may also be acetyl, or a
pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable carrier.
57. A method for treating or preventing radiation-induced cellular
damage or sunlight-induced cellular damage comprising administering
to an animal an effective amount of a composition comprising an
acyl derivative of 2'-deoxyguanosine having the formula 13wherein
R.sub.1, R.sub.2, and R.sub.3 are the same or different and each is
hydrogen or an acyl group derived from (a) an unbranched fatty acid
with 3 to 22 carbon atoms, (b) an amino acid selected from the
group consisting of glycine, the L forms of alanine, valine,
leucine, isoleucine, tyrosine, proline, hydroxyproline, serine,
threonine, cysteine, aspartic acid, glutamic acid, arginine,
lysine, histidine, phenylalanine, carnitine, and ornithine, (c)
nicotinic acid, or (d) a dicarboxylic acid having 3 to 22 carbon
atoms, provided that not all of R.sub.1, R.sub.2, and R.sub.3 are
H, and where R.sub.3 is not H, then R.sub.1 and/or R.sub.2 may also
be acetyl, or a pharmaceutically acceptable salt thereof, and a
pharmaceutically acceptable carrier.
58. A method for treating or preventing radiation-induced cellular
damage or sunlight-induced cellular damage comprising administering
to an animal an effective amount of a composition comprising an
acyl derivative of 2'-deoxycytidine, having the formula 14wherein
R.sub.1, R.sub.2, and R.sub.3 are the same or different and each is
hydrogen or an acyl group derived from (a) an unbranched fatty acid
with 3 to 22 carbon atoms, (b) an amino acid selected from the
group consisting of glycine, the L forms of alanine, valine,
leucine, isoleucine, tyrosine, proline, hydroxyproline, serine,
threonine, cysteine, aspartic acid, glutamic acid, arginine,
lysine, histidine, carnitine, and ornithine, (c) nicotinic acid, or
(d) a dicarboxylic acid having 3 to 22 carbon atoms, provided that
not all of R.sub.1, R.sub.2, and R.sub.3 are H, and where R.sub.3
is not H, then R.sub.1 and/or R.sub.2 may also be acetyl, or a
pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable carrier.
59. A method for treating or preventing radiation-induced cellular
damage or sunlight-induced cellular damage comprising administering
to an animal an effective amount of a composition comprising an
acyl derivative of 2'-deoxythymidine, having the formula 15wherein
R.sub.1 is an acyl group derived from (a) an unbranched fatty acid
with 3 to 15 or 17 to 22 carbon atoms, (b) an amino acid selected
from the group consisting of glycine, the L forms of alanine,
valine, leucine, isoleucine, tyrosine, proline, hydroxyproline,
serine, threonine, cysteine, aspartic acid, glutamic acid,
arginine, lysine, histidine, carnitine, and ornithine, (c)
nicotinic acid, or (d) a dicarboxylic acid having 3 to 22 carbon
atoms, and R.sub.2 and R.sub.3 are H, or a pharmaceutically
acceptable salt thereof, and a pharmaceutically acceptable
carrier.
60. A method for treating or preventing radiation-induced cellular
damage or sunlight-induced cellular damage comprising administering
to an animal an effective amount of a composition comprising an
acyl derivative of 2'-deoxythymidine, having the formula 16wherein
R.sub.1 is H, R.sub.2 is an acyl group derived from (a) an
unbranched fatty acid with 3 to 13 or 15 to 22 carbon atoms, (b) an
amino acid selected from the group consisting of glycine, the L
forms of alanine, valine, leucine, isoleucine, tyrosine, proline,
hydroxyproline, serine, threonine, cysteine, aspartic acid,
glutamic acid, arginine, lysine, histidine, carnitine, and
ornithine, (c) nicotinic acid, or (d) a dicarboxylic acid with 3 to
22 carbon atoms, and R.sub.3 is H or a pharmaceutically acceptable
salt thereof, and a pharmaceutically acceptable carrier.
61. A method for treating or preventing radiation-induced cellular
damage or sunlight-induced cellular damage comprising administering
to an animal an effective amount of a composition comprising an
acyl derivative of 2'-deoxythymidine, having the formula 17wherein
R.sub.1 and R.sub.2 are the same or different and each is an acyl
group derived from (a) an unbranched fatty acid with 5 to 22 carbon
atoms, (b) an amino acid selected from the group consisting of
glycine, the L forms of alanine, valine, leucine, isoleucine,
tyrosine, proline, hydroxyproline, serine, threonine, cysteine,
aspartic acid, glutamic acid, arginine, lysine, histidine,
carnitine, and ornithine, (c) nicotinic acid, or (d) a dicarboxylic
acid with 3 to 22 carbon atoms, and R.sub.3 is H or a
pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable carrier.
62. A method for treating or preventing radiation-induced cellular
damage or sunlight-induced cellular damage comprising administering
to an animal an effective amount of a composition comprising an
acyl derivative of 2'-deoxythymidine, having the formula 18wherein
R.sub.1 and R.sub.2 are the same or different and each is an acyl
group derived from (a) an unbranched fatty acid with 2 to 22 carbon
atoms, (b) an amino acid selected from the group consisting of
glycine, the L forms of alanine, valine, leucine, isoleucine,
tyrosine, proline, hydroxyproline, serine, threonine, cysteine,
aspartic acid, glutamic acid, arginine, lysine, histidine,
carnitine, and ornithine, (c) nicotinic acid or (d) a dicarboxylic
acid with 3 to 22 carbon atoms, and R.sub.3 is an acyl group
derived from an optionally substituted benzoyl or heterocyclic
carboxylic acid that is substantially nontoxic, or a
pharmaceutically acceptable salt thereof, and a pharmaceutically
acceptable carrier.
63. A method for treating or preventing radiation-induced cellular
damage or sunlight-induced cellular damage comprising administering
to an animal an effective amount of a composition comprising an
effective amount of each of at least two compounds selected from at
least two of the groups of compounds having formulae 19wherein
R.sub.1, R.sub.2, and R.sub.3 are the same or different and each is
H or an acyl group derived from a carboxylic acid, provided that at
least one of said substituents R.sub.1, R.sub.2, and R.sub.3 on
each of said groups of compounds is not hydrogen, or
pharmaceutically acceptable salts thereof, and a pharmaceutically
acceptable carrier.
Description
FIELD OF THE INVENTION
[0001] This invention relates generally to acyl derivatives of
deoxyribonucleosides and to the use of those derivatives to enhance
the delivery of exogenous deoxyribonucleosides to animal tissue.
More specifically, this invention relates to the acyl derivatives
of 2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxycytidine and
2'-deoxythymidine and the use of those novel derivatives to
increase the bioavailability of the deoxyribonucleosides to animal
tissue and thereby to support cellular metabolic functions. Even
more specifically, this invention relates to the use of the novel
acyl derivatives to treat or prevent a variety of physiological and
pathological conditions in cell tissue, including damage by
radiation, sunlight, mutagens, wounds, and other conditions.
BACKGROUND OF THE INVENTION
[0002] There are many physiological and pathological conditions of
animal tissue where the supply of exogenous deoxyribonucleosides
may have useful therapeutic applications. In the treatment of
wounds, repair of liver tissue, promotion of repair and survival
after radiation, and numerous other conditions, the supply of DNA
and/or deoxyribonucleosides at a high and sustained level may
substantially improve the natural DNA and tissue repair processes
of the affected cells.
[0003] In promoting wound healing, liver regeneration, recovery
from radiation damage, and in other pathological and physiological
conditions, it is likely that exogenously supplied DNA serves
merely as a storage depot for deoxyribonucleosides. That depot
gradually releases deoxyribonucleotides and deoxyribonucleosides
during enzymatic degradation. Thus the administration of
deoxyribonucleosides or derivatives disclosed herein may have value
as a method for delivering those deoxyribonucleosides to tissues,
which method is preferable to the administration of foreign DNA
insofar as wound healing, tissue regeneration, recovery from
irradiation, and the like, is concerned.
[0004] A number of investigators have attempted to use DNA and/or
deoxyribonucleosides to treat a variety of conditions in
experimental animals and to enhance or augment cellular repair
processes, including DNA repair. It has been demonstrated that
administration of exogenous DNA to experimental animals after
exposure to ionizing radiation can result in dramatically increased
survival and functional recovery. Studies on cell cultures in vitro
demonstrate that the actual restorative agents are probably
deoxyribonucleosides, the enzymatic degradation products of DNA.
These compounds enhance the actual repair of damaged DNA in vitro.
However, depolymerized DNA or deoxyribonucleosides administered to
animals were ineffective in promoting survival or recovery after
irradiation. Kanazir et al., Bull. Inst. Nuc. Sci "Boris, Kidrinch"
9:145-153 (1959). There is reason to believe that this apparent
contradiction is due to the rapid catabolism of
deoxyribonucleosides in vivo by the liver and other organs. Thus,
after administration of deoxyribonucleosides, tissues were only
exposed to effective concentrations for a matter of minutes. Beltz,
et al., Bioch. Biophys. Acta 297:258-267 (1973). In cell cultures,
optimum survival after irradiation was found when
deoxyribonucleosides were present in the incubation medium for at
least 3 hours. When DNA is administered by intraperitoneal
injection, it is gradually depolymerized to give a sustained
release of free deoxyribonucleosides into the circulation. DNA is
not, however, a suitable pharmaceutical agent to administer to
humans, either orally or parenterally.
[0005] Hunting, D. J., et al., Carcinogenesis 6:1525-1528 (1985),
disclose that deoxyribonucleotide synthesis is rate limiting for
excision repair of UV-induced DNA damage. The authors found that
there was an increase in repair ligation in cells made permeable to
added deoxyribonucleotide triphosphates.
[0006] Golba, S., et al., Int. J. Rad. Biol. 13:261-268 (1967),
disclose that after whole-body irradiation, administration of
heterologous DNA improved survival and accelerated the rate of
recovery of body weight and of red blood cells, granulocytes and
lymphocyte counts in the peripheral blood. No secondary disease or
change in the blood count was observed in the next 12 months. Goh,
K., Proc. Soc. Exp. Biol. Med. 145:938-943 (1974), discloses
addition of exogenous deoxyribonucleotides resulted in prevention
or healing of "pulverized" chromosomes found in cultures of
leukocytes taken from a human subjected to accidental exposure to
fast neutron and gamma irradiation. Horikawa, M., et al., Exp. Cell
Res. 34:198-200 (1964), disclose the effect of the addition of
various cell extracts and compounds to an incubation medium
containing mouse L cells in culture which were irradiated in
culture with X-irradiation (2000 R). Homogenates of L cells, L cell
nuclei, or purified DNA from either L cells or salmon sperm all
strongly enhanced the survival of the irradiated cells. RNA from
either yeast or L cells was found to be ineffective. The authors
suggest that the DNA hydrolysates (e.g., deoxynucleotides) are the
actual reactivating agents, since heterologous DNA is as effective
as homologous DNA.
[0007] Pantic, V., et al., Nature 193:993-994 (1962), disclose
administration of DNA to X-irradiated rats given lethal doses of
radiation. The authors found that while DNA treatment did not
totally prevent cellular damage in the intestine and liver after
irradiation, tissue structure and function were much closer to
normal in DNA-treated animals examined 4 or 9 days after
irradiation than in untreated irradiated controls.
[0008] Paoletti, C., et al., Rev. Francais. Etudes Clin. et Bio.
9:950-955 (1964), disclose a study on the effect of administration
of DNA and 2-aminoethyl-isothiouronium (AET) to rats. Mice were
given a mixture of AET and thiogel orally, then irradiated (700
rad) and subsequently given i.p. injections of 1 mg calf thymus
DNA. The mice receiving the DNA injections recovered their weight
and initial leukocyte counts more rapidly than mice similarly
treated but not receiving the DNA injections.
[0009] Petrovic, D., et al., Int. J. Radiat. Biol. 18:243-258
(1970), disclose evidence concerning the molecular basis of the
restorative effect of DNA in cultured mammalian cells. The authors
found that the survival of irradiated cells in culture was enhanced
by the addition of either DNA or equimolar amounts of
deoxyribonucleosides. DNA was effective only if serum containing
active deoxyribonuclease was present in the incubation medium.
Thus, the authors concluded that the deoxyribonucleosides were
probably the actual reactivating factors responsible for repair of
radiation-induced damage. In another study, Petrovic disclosed that
maximal restoration is attained when deoxyribnucleosides are in the
incubation medium for at least 3 hours after irradiation. The best
restoration was achieved with either a mixture of all four major
deoxyribonucleosides, or a combination of deoxyguanosine with
either deoxyadenosine or deoxycytidine. Petrovic, D., et al.,
Studia Biophysica 43:13-18 (1974). Petrovic et al. also report that
in irradiated HeLa cells, treatment with a mixture of the four
major deoxyribonucleosides increased survival. Petrovic et al.,
Int. J. Radiat. Res. 11:609-611 (1967).
[0010] Savkovic, N., Nature 203:1297-1298 (1964), discloses that 8
or 17 day old rats subjected to X-radiation (600 rem), and
immediately treated with homologous testes DNA, had a much higher
fertility rate than did untreated irradiated controls. Histological
studies demonstrated that DNA treatment after irradiation markedly
protected the structural integrity of the testes and the function
of the spermatogenic processes. Savkovic also reported that
heterologous DNA extracted from various organs of adult rats was
effective in enhancing the survival of mice subjected to
irradiation. The DNA reduced the effects of radiation by a factor
of 9 to 13. Savkovic, N., et al., Nature 211:1179-1180 (1966).
Savkovic, N., et al., Int. J. Rad. Biol. 9:361-368 (1965) also
disclose that treatment of irradiated rats with homologous DNA,
isolated from liver, thymus and spleen, increased survival and
fertility of the survivors. The death rate of the progeny of the
irradiated rats was strongly reduced in the case of animals that
received DNA after irradiation.
[0011] In another study, exposure of cultured calf liver cells to
X-radiation was found to cause chromosomal damage. When cells were
incubated with either DNA or equimolar concentration of
deoxyribonucleotides after irradiation, there was a marked
reduction in the incidence of chromosome damage. A mixture of dAMP
and dGMP was as effective as a mixture of all four major
deoxyribonucleotides. Ribonucleotides were ineffective in
preventing radiation-induced chromosome damage. Smets, L. A., et
al., Int. J. Rad. Biol. 13:269-273 (1967).
[0012] In a related study, administration of dCMP or dTMP to
irradiated mice was found to improve the restoration of
hematopoietic function. Soska, J., et al., Folia Biologica
5:190-198 (1959).
[0013] In another study of mice irradiated with gamma radiation,
administration of either a yeast RNA hydrolysate, an equimolar
mixture of 3'-nucleotides or a mixture of nucleosides resulted in a
significant prolongation of life span. However, long-term survival
was not enhanced. The nucleic acid derivatives were administered 30
minutes, 2 days, and 4 days after irradiation. The author observed
that the nucleosides, nucleotides, and RNA hydrolysate did not
increase the number of surviving stem cell colonies in spleen or
bone marrow, but rather appeared to improve the functional capacity
of irradiated cells during the critical period after irradiation.
These compounds also appeared to accelerate the process of
maturation and differentiation of the progeny of surviving stem
cells. Sugahara, T., et al., Brookhaven Symposia in Biology,
284-302 (1967).
[0014] In a study of guinea pigs subjected to X-radiation, animals
given RNA or ATP immediately before and after irradiation had much
higher 21-day survival rates than did untreated irradiated
controls. Most of the animals that survived the 21-day observation
period recovered fully, with no secondary radiation-induced
disease. Wagner, R., Int. J. Rad. Biol. 12:101-112 (1967).
[0015] In another study, administration of DNA from different
sources, including calf thymus, rat liver and spleen, herring and
salmon sperm, and Ehrlich ascites carcinoma cells was studied in
rats given lethal doses of gamma irradiation. All forms of DNA
significantly increased the survival of the irradiated rats. The
quantitative differences in the effects of the DNA from different
sources were directly related to the molecular weight. The authors
found a reduction in therapeutic efficiency which is proportional
to the reduction in molecular size upon DNA shearing. Wilczok, T.,
et al., Int. J. Rad. Biol. 9:201-211 (1965).
[0016] The incidence of chromosomal abnormalities in lymphocytes
from radiologists chronically exposed to X-rays, was determined
before, during, and after treatment with DNA and ATP. The basal
incidence of chromosomal damage was substantially higher than in
unexposed control subjects. Daily injection of DNA and ATP resulted
in 2 to 3 fold decreases in the frequency of chromosomal
abnormalities. Following discontinuation of treatment, the
incidence of chromosomal damage returned toward pretreatment
levels. (Goyanes-Villaescusa, Lancet II:575 (1973).
[0017] There have also been reports on the use of DNA preparations
to treat wounds. For example, Dumont, Ann. Surg. 150:799 (1959),
disclose that exogenous DNA, applied to experimental wounds in
rabbit ears, accelerated the growth of granulation tissue in the
wounds. A mixture of DNA plus deoxyribonuclease (the enzyme
primarily responsible for degradation of DNA) was more effective in
accelerating fibroplasia than either DNA or deoxyribonuclease
alone. The total amount of granulation tissue formed after
treatment with DNA was not greater than in untreated controls; the
onset and rate of its growth were however significantly
accelerated. The authors suggest that low polymer DNA fragments are
the actual active agents.
[0018] Nicolau et al., Der Hautartzt 17:512 (1966), disclose a
study on experimental skin wounds on the backs of rats which were
treated daily with a 1% solution of DNA in physiological saline.
The wounds treated with local application of DNA were cicatrized
within four to eight days; those treated only with physiological
saline were cicatrized only after 10 to 15 days.
[0019] Marshak et al., Proc. Soc. Exp. Biol. Med. 58:62 (1945),
disclose that application of DNA to experimental skin wounds in
rats resulted in a significant acceleration of the growth of
granulation tissue within the wounds, as compared to untreated
controls. Although the granulation tissue appeared sooner in
treated wounds, the final amount of granulation tissue was not
abnormal.
[0020] Newman et al., Am. J. Physiol. 164:251 (1951), disclose a
study of rats subjected to partial hepatectomy. The course of liver
regeneration was followed for 11 days. The livers of rats treated
with DNA regenerated significantly faster than livers in untreated
animals. RNA treatment also accelerated liver regeneration, though
not as markedly as DNA administration.
[0021] Certain derivatives of deoxyribonucleosides have been
prepared. Casida et al., Biochemical Pharmacology vol. 15, p.
627-644, 1966, describe the preparation of the 3'5'-diacetyl,
dipropionyl and dibutyryl esters of 2'-deoxythymidine. Rosowsky et
al., Cancer Treatment Reports vol. 65 No.1-2, p. 93-99,
January/February 1981, and Ensminger et al., Biochemical
Pharmacology vol. 28, p. 1541-1545, October 1978, describe the use
of thymidine 5'-O-pivaloate to supply thymidine to tissues.
[0022] Since the primary determinant of recovery or survival after
exposure to ionizing radiation or chemical mutagens is the
preservation or repair of DNA, a number of compounds have been
found which, when present in an organism at the time of exposure to
radiation or chemical mutagens, attenuate the damage to DNA and
other cellular structures. Included in this class of compounds are
antioxidants, sulfhydryl compounds, and the enzymes superoxide
dismutase and catalase. However, these compounds have been found to
be only moderately protective or practical to use in vivo, in part
because they can be toxic in effective concentrations. Since these
compounds must be present in the organism at the time of exposure
to radiation or chemical mutagens, they are obviously not useful in
the case of unexpected or accidental exposure.
[0023] Reportedly, sulfhydryl compounds are the most effective
radioprotective agents known. Examples of these compounds include
mercaptoethylamine (MEA), 2-8-amino-ethyl-isothiouronium-Br--HBr
(AET), 5-hydroxytryptamine (HT), and
5-2-(3-amino-propylamino)ethylphosphorothio- toic acid (WR-2721).
However, many of these compounds are toxic. Thus, several
investigators have attempted to increase. protection against
radiation damage and to decrease toxicity by using mixtures of
these chemical protectors. The results of these studies demonstrate
that the administration of mixtures of radioprotectors not only
increases the degree of protection for short and long term survival
compared with that from each substance given separately, but also
diminishes the toxicity of compounds such as AET or MEA.
Administration of sulfhydryl chemical radioprotectors before
exposure to radiation diminishes markedly the changes induced by
radiation in the structures. Maisin, J. R., in: Symposium on
Perspectives in Radioprotection, Armed Forces Radiobiology Research
Institute, Bethesda, Md., p. 53 (1987).
[0024] Thiols reportedly protect DNA by mechanisms comprising
hydroxyl radical scavenging and DNA radical repair mechanisms.
Thus, the extent of interactions of thiols with DNA determines the
amount of protection. Cationic thiols
(2-[(aminopropyl)amino]ethanethiol (WR-1065) and cysteamine) are
better protectors than neutral thiol (2-mercaptoethanol and
dithiothreitol) which are in turn better protectors than anionic
thiols (glutathione (GSH), 2-mercaptoethanesulfonic acid, and
mercaptosuccinate). Such differential binding provides a basis for
understanding why WR-1065, which scavenges hydroxyl radicals at a
rate comparable to that for GSH, effectively protects cells at
concentrations well below those of GSH. Fahery, R. C., ibid., p.
31.
[0025] In studies of Chinese hamster V-79 cells treated with gamma
radiation and with bromodeoxyuridine (BrdUrd) and light photolysis
were compared. When treated with gamma radiation, WR-2721 was found
to improve cell survival both by acting as a reducer of gamma
radiation, and by causing increase in DNA repair and increase in
rejoining of DNA strand breaks. Cysteamine has been shown to act as
a reducer of gamma radiation damage without affecting the rejoining
of strand breaks or DNA repair capacity. Nicotinamide (NA) has been
shown to directly affect DNA repair through the polyADPribose
system which is activated by DNA single strand breaks, thus
providing NA concentration dependent protection or sensitization.
These compounds exhibit a different effect on cells treated with
BrdUrd and light compared with gamma radiation. WR-2721 does not
reduce strand break formation. MEA and NA reduce damage formation
by about 30%. WR-2721 did not affect the rejoining of
BrdUrd/light-induced DNA strand breaks. Only NA increased the
repair capability of cells subjected to BrdUrd and light damage.
Prager, A., et al., ibid., p. 43.
[0026] In addition to the use of thiols, radioprotection has been
achieved with "biological response modifiers" (BRM), either alone
or in combination with other agents. Such biological response
modifiers include glucan, OK-432, Biostim, PSK, Lentinan,
Schizophyllan, Rhodexman, Levan, Mannozym, and MVE-2. Of these
BRM's, glucan was found to be the most radioprotective. Glucan is a
beta 1-3 polyglycan isolated from the yeast Saccharomyces
cerevisiae. Glucan's radioprotective capacity is attributed to its
ability both to protect and/or enhance recovery of hemoatopoietic
stem cell populations, and to enhance or maintain the function of
macrophage cell populations important in combatting otherwise
lethal post-irradiation opportunistic infections. The combination
of glucan and WR-2721 resulted in both additive and synergistic
radioprotective effects. Patchen, M. L., ibid., P. 68.
[0027] Other polysaccharides have also been found to be
radioprotective. Intravenous administration of the polysaccharide
extracted from the yeast Rhodotorula rubra, mannane mannozyme
(MMZ), and the particulate polyglucans GLP/B04 and GLP/B05
(unbranched glucans with alternating B-1,3 and B-1,6 bonds),
significantly decreased the mortality of mice exposed to a single
dose of X-rays. Maisin, J. R., ibid., p. 69.
[0028] The cytokines IL-2 and TNF have also been found to be
effective radioprotective compounds. Cytokines are released upon
administration of numerous inflammatory agents. Many of these
inflammatory agents stimulate the reticuloendothelial system and
are radioprotective. Neta, R., ibid, p. 71.
[0029] Thymic peptides, such as thymic factor TF-5, have also been
reported to reverse or greatly ameliorate immune depression due to
limited portal irradiation of thymus, circulating blood, and
lymphoid tissues. The immune restorative effect of thymic factors
is due to their maturational effect on bone marrow immunocyte
precursors. Chretien, P. B., ibid., p. 72.
[0030] The antioxidant enzymes glutathione peroxidase (GSH-Px),
superoxide dismutase (SOD), catalase, glutathione reductase and
glutathione transferase scavenge free radical species produced by
radiation and/or the products of free radical cellular damage, and
thus play a role in radioprotection. GSH-Px exhibits and best
correlation between enzyme activity and cell radiosensitivity.
Administration of enzyme preparations or drugs or chemicals which
mimic or activate or induce these enzymes may enhance
radioprotection. The radioprotectors MEA, WR-2721 and
diethyldithiocarbamate (DDC) enhance mouse liver GSH-Px activity 1
to 2 hours after administration. Selenium and selenium-containing
compounds also exhibit a small radioprotective effect. The levels
of GSH-Px in mouse bone marrow were found to increase 30% 24 hours
after administration of selenium. When selenium was administered
before WR-2721, a decrease in toxicity and an increase in
radioprotection was observed. Superoxide dismutase (SOD) and
catalase were also observed to increase upon administration of
selenium. In addition, metal ions and metal-containing compounds
which mimic antioxidant enzymes may also act as radioprotectors.
Copper and zinc metal ions in SOD are marginally radioprotective.
Mimetics of SOD include bis (3,5-diisopropylsalicylato) copper and
the bivalent copper complex of 3-mercapto-2-hydroxypropylether of
dextran. Dumar, K. S., ibid., p. 89. For a review on SOD, see
Fridovich, I., Annu. Rev. Biochem. 44: 147-159 (1975).
[0031] Induction of metallothionein (MT) in the body by treatment
with some heavy metals or immunostimulants has been found to be a
potent means for inducing radioprotection. The metal salts
CdCl.sub.2, MnCl.sub.2 or zinc acetate or the immunostimulants
OK-432 or IL-1 elevates MT levels in the liver of pretreated mice
10 to 20 times of the control level. The number of leukocytes as
well as erythrocytes were reduced temporarily even in pretreated
mice. However, the cell counts of pretreated mice showed a faster
recovery. Matsubara, J., et al., ibid., p. 99.
[0032] Vitamin A and beta carotene have also been suggested as
radioprotective agents. They may be involved in ameliorating the
oxidative damage in tissues of irradiated mammals which results
from production of free radicals such as hydroxy radicals or
H.sub.2O.sup.+ and its daughter products. Radiation may also create
injury to cell structures either by the direct effect of radiation
or by the production of toxic metabolites. Radiation injury results
in disturbed extracellular and intracellular oxygen levels and
perturbed intracellular electron transport and metabolism.
Oxidative damage may be enhanced by sudden elevations of local
oxygen levels caused by reperfusion of tissues after
radiation-induced vasoconstriction or by reversal of
radiation-induced bronchoconstriction. Damage occurs where local
oxygen levels are in excess of what the tissues can consume.
Vitamin A and beta carotene were found to exhibit protective action
in rodents exposed to whole body and local radiation. Seifter, E.,
et al., ibid. p. 104.
[0033] Prostaglandins and related compounds of the arachidonic acid
cascade protect cells in vivo from some degree of ionizing
radiation injury. Among the array of physiological actions of
prostaglandins is the protection of cells and tissues from a
variety of injuries including strong acids, bases, and absolute
ethanol. Prostaglandins were found to exhibit maximal protection at
levels a thousandfold lower than those needed for WR-2721. Hanson,
W. R., ibid., p. 105.
[0034] Methylene blue, a compound used clinically as an
anti-inflammatory, antimalarial, and antibacterial agent as well as
in the treatment of carbon monoxide poisoning and as an antidote
for cyanide poisoning, as also found to protect the intestinal
mucosa of rats subjected to sublethal radiation-induced damage.
Irradiation damages tissues through the production of highly
bioactive free radical species. Therefore, it was hypothesized that
methylene blue would also protect irradiated rats from free-radical
mediated tissue damage. Scheving, L. E., et al., ibid., p. 115.
[0035] In addition, N-arylacetyldehydroalanines reportedly inhibit
superoxide anion and hydroxyl free radical-mediated processes,
thereby providing radioprotective activity. Buc-Calderone, P., et
al., ibid., p. 116.
OBJECTS OF THE INVENTION
[0036] While the strategy of delivering DNA and/or
deoxyribonucleosides to physiologically or pathologically damaged
tissue has been recognized, the art has heretofore failed to
provide satisfactory methods for introducing deoxyribonucleosides
in sufficiently high and reliable amounts to successfully treat the
pathological and physiological conditions and to promote cellular
repair and survival of the animal. Moreover, although a variety of
compounds have been developed which protect animals against some
effects of ionizing radiation or chemical mutagens,
deoxyribonucleosides provided to tissues for a sufficient time have
the greatest clinical potential for post-exposure treatment of such
damage. Clinical implementation of this strategy, however, awaits
development of satisfactory and convenient methods for delivering
adequate quantities of deoxyribunucleosides to tissues in vivo.
Similarly, full appreciation and clinical implementation of the
capacity of deoxyribonucleosides to promote wound healing or tissue
repair awaits development of satisfactory methods for their
delivery to tissues in vivo.
[0037] It is thus a primary object of this invention to identify
pharmaceutically acceptable compounds which can efficiently be used
to deliver pharmacologically effective amounts of
deoxyribonucleosides or their respective derivatives to animal
tissue.
[0038] It is still a further object of this invention to provide a
family of deoxyribonucleoside derivatives which can be effectively
administered orally or parenterally, which have no undesirable
toxic effects, and which can be administered to animals and humans
to effectively promote cellular repair in a number of physiological
and pathological conditions and to promote survival of the animal
when administered after exposure to radiation has occurred.
[0039] It is still a further and related object of this invention
to provide certain derivatives of 2'-deoxyadenosine,
2'-deoxyguanosine, 2'-deoxycytidine, and 2'-deoxythymidine which,
when administered to an animal, enhance the bioavailability of
those deoxyribonucleosides to the animal tissue.
[0040] It is a related object of this invention to substantially
improve the bioavailability of 2'-deoxyadenosine,
2'-deoxyguanosine, 2'-deoxycytidine, and 2'-deoxythymidine by
enhancing the transport of these deoxyribonucleosides across the
gastrointestinal tract, the blood-brain barrier, and other
biological membranes.
[0041] It is still a further and more specific object of this
invention to provide a family of deoxyribonucleoside derivatives
for the treatment of a variety of heart, muscle, liver, bone, skin,
and other pathological and physiological conditions.
[0042] It is still a further object of this invention to provide
deoxyribonucleoside derivatives and methods for using those
derivatives which are safe, inexpensive, and accelerate the normal
cellular processes of regeneration and healing.
SUMMARY OF THE INVENTION
[0043] These and other objects of the invention are achieved by the
administration of certain acyl derivatives of 2'-deoxyadenosine,
2'-deoxyguanosine, 2'-deoxycytidine, and 2'-deoxythymidine. These
acyl derivatives can be used to prevent or treat radiation,
sunlight and mutagen-induced cellular damage, to improve the
healing of wounds, or repair damaged tissues, and in the treatment
of other physiological and pathological tissue conditions.
[0044] Broadly, the acyl derivatives of 2'-deoxyadenosine are those
having the formula 1
[0045] wherein R is hydrogen or an acyl radical of a metabolite
other than acetyl, with the proviso that at least one R is not
hydrogen, or the pharmaceutically acceptable salt thereof.
[0046] The preferred acyl derivatives of 2'-deoxyadenosine are
those having the formula 2
[0047] wherein R is H or an acyl group derived from a carboxylic
acid selected from one or more of the group consisting of pyruvic
acid, lactic acid, enolpyruvic acid, an amino acid, a fatty acid
other than acetic acid, lipoic acid, nicotinic acid, pantothenic
acid, succinic acid, fumaric acid, p-aminobenzoic acid,
betahydroxybutyric acid, orotic acid, and carnitine, with the
proviso that at least one R is not hydrogen, or the
pharmaceutically acceptable salt thereof.
[0048] Broadly, the acyl derivatives of 2'-deoxyguanosine are those
having the formula 3
[0049] wherein R is hydrogen or an acyl radical of a metabolite
other than acetyl, with the proviso that at least one R is not
hydrogen, or the pharmaceutically acceptable salt thereof.
[0050] The preferred acyl derivatives of 2'-deoxyguanosine are
those having the formula 4
[0051] wherein R is H or an acyl group derived from a carboxylic
acid selected from one or more of the group consisting of pyruvic
acid, lactic acid, enolpyruvic acid, an amino acid, a fatty acid
other than acetic acid, lipoic acid, nicotinic acid, pantothenic
acid, succinic acid, fumaric acid, p-aminobenzoic acid,
betahydroxybutyric acid, orotic acid, and carnitine, with the
proviso that at least one R is not hydrogen, or the
pharmaceutically acceptable salt thereof.
[0052] Broadly, the acyl derivatives of 2'-deoxycytidine are those
having the formula 5
[0053] wherein R is hydrogen or an acyl radical of a metabolite
other than acetyl, with the proviso that at least one R is not
hydrogen, or the pharmaceutically acceptable salt thereof.
[0054] The preferred acyl derivatives of 2'-deoxycytidine are those
having the formula 6
[0055] wherein R is H or an acyl group derived from a carboxylic
acid selected from one or more of the group consisting of pyruvic
acid, lactic acid, enolpyruvic acid, an amino acid, a fatty acid
other than acetic acid, lipoic acid, nicotinic acid, pantothenic
acid, succinic acid, fumaric acid, p-aminobenzoic acid,
betahydroxybutyric acid, orotic acid, and carnitine, with the
proviso that at least one R is not hydrogen, or the
pharmaceutically acceptable salt thereof.
[0056] Broadly, the acyl derivatives of 2'-deoxythymidine are those
having the formula 7
[0057] wherein R is hydrogen or an acyl radical of a metabolite
other than a fatty acid having less than five carbon atoms, with
the proviso that at least one R is not hydrogen, or the
pharmaceutically acceptable salt thereof.
[0058] The preferred acyl derivatives of 2'-deoxythymidine are
those having the formula 8
[0059] wherein R is H or an acyl group derived from a carboxylic
acid selected from one or more of the group consisting of pyruvic
acid, lactic acid, enolpyruvic acid, an amino acid, a fatty acid
containing 5 or more carbon atoms, lipoic acid, nicotinic acid,
pantothenic acid, succinic acid, fumaric acid, p-aminobenzoic acid,
betahydroxybutyric acid, orotic acid and carnitine, with the
proviso that at least one R substituent is not hydrogen, or the
pharmaceutically acceptable salt thereof.
[0060] The acyl derivatives of 2'-deoxythymidine may also be those
having the formula 9
[0061] wherein R" is hydrogen or an acyl radical of a metabolite,
with the proviso that the R" on nitrogen is not hydrogen, or the
pharmaceutically acceptable salt thereof.
[0062] Preferred acyl derivatives of 2'-deoxythymidine are those
having the formula 10
[0063] wherein R" is H or an acyl group derived from a carboxylic
acid selected from one or more of the group consisting of pyruvic
acid, lactic acid, enolpyruvic acid, an amino acid, a fatty acid,
lipoic acid, nicotinic acid, pantothenic acid, succinic acid,
fumaric acid, p-aminobenzoic acid, betahydroxybutyric acid, orotic
acid, and carnitine, with the proviso that the R" on nitrogen is
not hydrogen, or the pharmaceutically salt thereof.
[0064] The invention also includes compounds having formulae I-IV
wherein the ribose moiety is monoacylated at the 3' or 5' position
with the derivative of a fatty acid and includes 3',5'diacylated
derivatives of compounds I-IV wherein at least one such substituent
is derived from a fatty acid having 5 or more carbon atoms.
[0065] The acyl derivatives of 2'-deoxyadenosine,
2'-deoxyguanosine, 2'-deoxycytidine, and 2'-deoxythymidine having
formulae I, II, III, and V, desirably are substituted with an acyl
derivative of a carboxylic acid having 3-22 carbon atoms.
[0066] Where acyl derivatives of any of the compounds of formulae
I-V are substituted by an acyl group derived from. an amino acid,
the amino acid is desirably selected from the group consisting of
glycine, the L forms of alanine, valine, leucine, isoleucine,
phenylalanine, tyrosine, proline, hydroxyproline, serine,
threonine, cysteine, cystine, methionine, tryptophan, aspartic
acid, glutamic acid, arginine, lysine, histidine, ornithine, and
hydroxylysine.
[0067] In a preferred embodiment of the invention, a mixture of at
least two acyl derivatives of 2'-deoxyadenosine, 2'-deoxyguanosine,
2'-deoxycytidine, and 2'-deoxythymidine is used. Said compositions
contain at least two of the acyl derivatives having the formulae
11
[0068] wherein R'" is H or an acyl group derived from a carboxylic
acid selected from one or more of the group consisting of pyruvic
acid, lactic acid, enolpyruvic acid, an amino acid, a fatty acid,
lipoic acid, nicotinic acid, pantothenic acid, succinic acid,
fumaric acid, p-aminobenzoic acid, betahydroxybutyric acid, orotic
acid, and carnitine, with the proviso that at least one R is not
hydrogen, or the pharmaceutically acceptable salt thereof.
[0069] Further substantial benefits may be obtained, particularly
where the compositions of the invention are used to ameliorate the
effects of radiation, if a radioprotective compound is included
together with one or more of the acyl deoxyribonucleosides. The
radioprotective compounds may be those selected from the group
consisting of WR-2721, NAC, DDC, cysteamine, 2-mercaptoethanol,
mercaptoethylamine dithiothreitol, glutathione,
2-mercaptoethanesulfonic acid, WR-1065, nicotinamine,
5-hydroxytryptamine, 2- -aminoethyl-isothiouronium-Br--Hbr,
glucans, GLP/B04, GLP/B05, OK-432, Biostim, PSK, Lentinan,
Schizophyllan, Rhodexman, Levan, Mannozym, MVE-2, MNR, MMZ, IL-1,
TNF, thymic factor TF-5, glutathione peroxidase, superoxide
dismutase, catalase, glutathione reductase, glutathione
transferase, selenium, CdCl.sub.2, MnCl.sub.2, Zn acetate, Vitamin
A, beta carotene, prostaglandins, tocopherol, methylene blue and
PABA.
[0070] The invention is also embodied in pharmaceutical
compositions which comprise one or more of the novel
deoxyribonucleosides together with a pharmaceutically acceptable
carrier. In addition, known acetyl derivatives of the
2'-deoxyadenosine, 2'-deoxyguanosine, 2'-deoxy-cytidine and
2'-deoxythymidine as well as the fatty acid derivatives of
thymidine wherein the acyl group contains 3 or 4 carbon atoms may
be used alone, in combination with one another or in combination
with one or more novel compounds, in pharmaceutical compositions of
the invention. The composition may further include a
radioprotective compound as described. The compositions may be in
the form of a liquid, a suspension, a tablet, a dragee, an
injectable solution, a topical solution, or a suppository.
[0071] A skin lotion may be advantageously prepared by combining an
effective amount of one or more of the acyl deoxyribonucleosides of
the invention together with a suitable carrier. Such a skin lotion
advantageously contains from 0.1 to 5 percent by weight of the
deoxyribonucleosides and, if desirable, the radioprotective
compound.
[0072] The pharmaceutical compositions of the invention can also be
embodied in bioerodible microcapsules, the microcapsules desirably
being selected from the group consisting of polylactate or
lactate-glycolate copolymers.
[0073] It is believed that the delivery of exogenous
deoxyribonucleosides to the tissue of an animal can be effectively
achieved by administering to that animal an effective amount of an
acyl derivative of a deoxyribonucleoside of formulae I-V. By
enhancing the delivery of exogenous deoxyribonucleosides, and
thereby increasing their bioavailability, it may be possible to
treat physiological or pathological conditions of the tissues of an
animal by essentially supporting the metabolic functions thereof.
Without being bound by theory, the invention may work, as well, by
increasing the bioavailability of nucleoside anabolites e.g.
nucleotides or nucleotide-derived cofactors. Administration of the
nucleosides per se increases their bioavailability but, due to
rapid catabolism, this may not result in significant elevation of
nucleotide levels; i.e., one does not necessarily get an increase
in intracellular levels because at lower nucleoside levels there is
rapid uptake by the cells whereas at higher levels there is
saturation and the excess is degraded. The invention is believed to
work by delivering a steady supply of nucleoside at low levels.
[0074] It is believed that the novel compounds and compositions of
the invention may be used advantageously in methods for treating
cardiac insufficiency, myocardial infarction, the consequences of
hypertension, cirrhosis of the liver, diabetes, senescence, adrenal
insufficiency, the complications of pregnancy, cerebrovascular
disorders, senile dementias, Parkinson's disease, demyelinating
disorders, cerebellar ataxia, infant respiratory distress syndrome,
and lung disorders, or to enhance bone healing or muscle
performance.
[0075] The specific methods where advantages may be achieved using
the compounds and compositions of the invention include treating or
preventing radiation-induced cellular damage, preventing
sunlight-induced cellular damage, ameliorating the effects of
aging, preventing mutagen-induced cellular damage, healing damaged
tissue, healing skin wounds, healing burn tissue, healing diseased
or damaged liver tissue, healing heart muscle damaged as a result
of myocardial infarction, treating damaged bone marrow, and
enhancing erythropoiesis. In treating all of these conditions, a
compound of the invention, with or without additional carriers,
radioprotective compounds, and other adjuvants, are administered to
an animal.
[0076] The invention essentially enhances the transport of
deoxyribonucleosides across biological membranes, including the
gastrointestinal tract (i.e., transport from the gut into the
bloodstream) and the blood-brain barrier. The rapid catabolism by
nucleoside phosphorylases or nucleoside deaminases is also
substantially prevented.
[0077] Administration of the acylated derivatives offers certain
advantages over the nonderivatized compounds. The acyl substituents
can be selected to increase the lipophilicity of the nucleoside,
thus improving its transport from the gastrointestinal tract into
the bloodstream. The acylated derivatives are effective when
administered orally. The acylated derivatives are resistant to
catabolism by nucleoside deaminases and nucleoside phosphorylases
in the intestine, liver, other organs, and the bloodstream. Thus,
administration of the acylated derivatives of the invention, either
orally or parenterally, allows sustained delivery of high levels of
deoxyribonucleosides to the tissues of an animal.
DESCRIPTION OF THE DRAWINGS
[0078] FIG. 1 is a graph illustrating the rates of degradation of
deoxyribonucleosides in plasma. The following abbreviations were
used:
[0079] dT=2'-Deoxythymidine
[0080] dC=2'-Deoxycytidine
[0081] dG=2'-Deoxyguanosine
[0082] dA=2'-Deoxyadenosine.
[0083] FIG. 2 is a graph illustrating the disappearance of
deoxyadenosine derivatives in plasma.
[0084] FIG. 3 is a graph illustrating the disappearance of
dexoyadenosine derivatives in liver extract.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0085] A "metabolite" is a chemical compound that is formed by, or
participates in, a metabolic reaction. In the context of this
application, metabolites include not only acyl substituents known
to be synthesized within the human body, but also naturally
occurring (but perhaps synthesized rather than extracted)
substituents that might be derived from other animal or plant
sources. The limiting criteria are that the compound should be
substantially nontoxic and biocompatible, and should readily enter
into metabolic pathways in vivo, so as to present essentially no
toxicity during long-term consumption in the doses proposed. It is
preferable that the substituents be metabolized rather than
excreted intact (or conjugated through detoxification reactions),
as concentration of carboxylic acids within the kidney may lead to
undesirable excessive acidity. Therefore, carboxylic acids that
normally or easily participate in intermediary, catabolic, or
anabolic metabolism are preferred substituents.
[0086] The term "pharmaceutically acceptable salts" means salts
with pharmaceutically acceptable acid addition salts of the
deoxyribonucleoside derivatives, which include, but are not limited
to, sulfuric, hydrochloric, or phosphoric acids.
[0087] The term "coadministered" means that at least two of the
acylated derivatives of the invention are administered during a
time frame wherein the respective periods of pharmacological
activity overlap.
[0088] "Acyl derivatives" means derivatives of a
2'-deoxyribonucleoside in which a substantially nontoxic organic
acyl substituent derived from a carboxylic acid is attached to one
or more of the free hydroxyl groups of the ribose moiety of the
deoxyribonucleoside with an ester linkage and/or where such a
substituent is attached to a primary or secondary amine in the
pyrimidine ring of deoxycytidine or deoxythymidine, or in the
purine ring of deoxyadenosine or deoxyguanosine, with an amide
linkage. Such acyl substituents are derived from carboxylic acids
which include, but are not limited to, compounds from the group
consisting of pyruvic acid, lactic acid, enolpyruvic acid, an amino
acid, a fatty acid, lipoic acid, nicotinic acid, pantothenic acid,
succinic acid, fumaric acid, p-aminobenzoic acid,
betahydroxybutyric acid, orotic acid, and carnitinic acid.
Preferred acyl substituents are compounds which are normally
present in the body, either as dietary constituents or as
intermediary metabolites, which are essentially nontoxic when
cleaved from the deoxyribonucleoside in vivo.
[0089] "Amino acids" include, but are not limited to, glycine, the
L forms of alanine, valine, leucine, isoleucine, phenylalanine,
tyrosine, proline, hydroxyproline, serine, threonine, cysteine,
cystine, methionine, tryptophan, aspartic acid, glutamic acid,
arginine, lysine, histidine, ornithine, hydroxylysine, and other
naturally occuring amino acids.
[0090] "Fatty acids" are carboxylic acids having 2-22 carbon atoms.
Such fatty acids may be saturated, partially saturated or
polyunsaturated.
[0091] Preferred acyl derivatives of 2-deoxyribonucleosides for
enhancing transport across biological membranes are those which are
more lipophilic than are the parent nucleosides. In general,
lipophilic acyl nucleoside derivatives have acyl substituents which
are nonpolar (aside from the carboxylate group). Lipophilic acyl
substituents include, but are not limited to, groups derived from
acetic acid, lipoic acid, and fatty acids. One of ordinary skill in
the art can determine whether a particular acyl-substituted
nucleoside derivative is more lipophilic than the underivatized
nucleoside using standard techniques, i.e., comparison of the
partition coefficients determined in water-octanol mixtures.
[0092] Following passage of the acylated nucleoside derivative from
the gastrointestinal tract into the bloodstream, across the
blood-brain barrier, or across other biological membranes, the acyl
substituents are cleaved by plasma and tissue esterases (or
amidases) to give the free nucleosides. The preferred acyl groups
of the invention are naturally occurring metabolites in the body,
or are compounds which readily enter intermediary metabolic
pathways. Thus they offer little toxicity when released in vivo by
endogenous esterases or amidases.
[0093] The rate of removal of the acyl substituents in vivo is a
function of the specificity of plasma and tissue deacylating
enzymes (primarily esterases or amidases). Fatty acid substituents
containing 4 to 8 carbon atoms are cleaved much more rapidly in
vivo than are fatty acids with either more or fewer carbon atoms.
Acyl substituents attached to an amine group in the pyrimidine ring
of deoxycytidine or deoxythymidine, or the purine ring of
deoxyadenosine or deoxyguanosine, with an amide linkage are cleaved
more slowly than are substituents attached to hydroxl groups of
ribose with an ester linkage.
[0094] It is also possible to prepare acyl nucleoside derivatives
which contain both polar and nonpolar acyl substituents. The polar
acyl group will retard passage of the nucleoside derivative from
the gastrointestinal tract, allowing for a more sustained delivery
of the compound into the bloodstream after a single dose. The polar
group may be cleaved by esterases, amidases, or peptidases present
in the intestinal tract to give a nucleoside with a nonpolar acyl
substituent which may then efficiently enter the circulation. Polar
acyl substituents may be chosen by one of ordinary skill in the
art, without undue experimentation, which are cleaved at a slower
rate than are nonpolar acyl substituents.
[0095] The acyl derivatives are also less susceptible to
degradation of the nucleoside moiety by enzymes in plasma and
non-target tissues, and are also less susceptible to elimination
from the bloodstream via the kidneys. For parenteral injection,
acyl derivatives with polar substituents, which are therefore water
soluble yet resistant to premature degradation or elimination, may
be used with advantage. Preferred acyl substituents for such
applications include those derived from glycolic and lactic acids
and from amino acids with polar side chains.
[0096] Therapeutic Uses
[0097] The lipophilic acyl deoxyribonucleoside derivatives of the
invention are useful for enhancing the transport of the
deoxyribonucleosides across biological membranes including the
gastrointestinal tract and blood-brain barrier in animals and
thereby increase the bioavailability of the deoxyribonucleosides.
Foremost among such animals are humans; however, the invention is
not intended to be so limiting, it being within the contemplation
of the invention to treat all animals which may experience a
beneficial effect from the administration of the acyl
deoxyribonucleosides of the invention.
[0098] The compositions of the present invention may be
administered to an animal either before or after exposure to
radiation, sunlight or mutagens. The acyl derivative form of the
deoxyribonucleosides provides an orally effective means for
delivery of deoxyribonucleosides to tissues. These derivatives may
also be given parenterally. Administration of the derivatives
avoids the problem of rapid catabolism by gastrointestinal, liver
and plasma enzymes.
[0099] As shown in FIG. 1, free deoxyguanosine (dG) and
deoxyadenosine (dA) are degraded in plasma at a much higher rate
than deoxythymidine (dT) and deoxycytidine (dC). Thus, the
compositions of the invention may include levels of dG and dA which
are higher than the levels of dT and dC to compensate for this
differential rate of degradation.
[0100] Acyl substituents for derivatization of dA and dG may be
selected which are slowly cleaved by esterases so that a prolonged
bioavailability of these deoxyribonucleosides may be maintained.
Since dT and dC are more slowly degraded, an acyl substituent which
is more rapidly cleaved may be used.
[0101] The fates of deoxyadenosine, N4-acetyldeoxyadenosine, and
N4-valeryldeoxyadenosine in plasma are shown in FIG. 2. Each of
these compounds was added to separate aliquots of rat plasma, at
initial concentrations of 20 micromolar. The plasma was sampled at
various time points, and the desired compounds were assayed by
liquid chromatography.
[0102] Deoxyadenosine (dA) is very rapidly degraded in plasma,
disappearing within 10 minutes. Administration of this compound to
an animal or human subject would make deoxyadenosine available to
tissues for a very short period of time.
[0103] N4-acetyldeoxyadenosine and N4-valeryldeoxyadenosine are,
however, deacylated in plasma (to form deoxyadenosine) over a
period of several hours. Therefore, administration of either of
these compounds would result in prolonged availability of
deoxyadenosine to tissues.
[0104] The fates of deoxyadenosine, N4-acetyldeoxyadenosine, and
N4-valeryldeoxyadenosine in liver extract are shown in FIG. 3. Each
of these compounds was added to separate aliquots of an aqueous
extract of rat liver, at initial concentrations of 20 micromolar.
The extract was sampled at various time points, and the desired
compounds were assayed by liquid chromatography.
[0105] Deoxyadenosine (dA) is extremely rapidly degraded in plasma,
disappearing within 1 minute. The initial degradation product is
deoxyinosine, which is not directly reutilizable by tissues.
Administration of deoxyadenosine per se to an animal or human
subject would make deoxyadenosine available to tissues for only a
very short period of time.
[0106] N4-acetyldeoxyadenosine and N4-valeryldeoxyadenosine are,
however, deacylated in liver extract (to form deoxyadenosine) over
a period of more than 1 hour. Therefore, administration of either
of these compounds would result in prolonged availability of
deoxyadenosine to liver or other organs.
[0107] Thus a mixture of several different acyl derivatives of each
deoxyribonucleoside in an administered dose may be selected to
provide optimal bioavailability. A composition containing
3',5'-diacetyl-2'-deoxy- cytidine, 5'-palmitoyl-2'-deoxycytidine,
and N.sup.4-palmitoyl-2'-deoxycyt- idine (and corresponding
derivatives of other deoxyribonucleosides) provides a more
prolonged bioavailability of nucleosides after a single dose than
does an administration of a single acyl derivative of each
nucleoside. Thus, after administration of the mixture described
above, the acetylated compound is relatively rapidly deacetylated,
yielding free deoxycytidine (or other desired deoxyribonucleosides)
shortly after administration. The 5'-palmitoyl derivative is
deacylated more slowly, providing additional free deoxycytidine
after the deoxycytidine derived from
3',5'-diacetyl-2'-deoxycytidine has been metabolized by tissues.
The N.sup.4-palmitoyl derivative is deacylated still more slowly
than is 5'-palmitoyl-2'-deoxycytidine.
[0108] The acyl deoxyribonucleoside compositions of the invention
also find use in treatment of ultraviolet light-induced cellular
damage that occurs upon exposure to sunlight. Within 72 hours after
exposure to sunlight, there is an increase in the number of
epidermal cells and a high rate of mitotic activity. The rate of
cell proliferation decreases after 7 to 10 days and the thickness
of the epidermis gradually returns to normal within the next 30 to
60 days. Damage to DNA by sunburn-producing UV light (290-320 nm)
may result in mutation or cell death. The principal photoproducts
are pyrimidine dimers (e.g., thymine dimers). Cell membranes, DNA,
RNA, protein and other molecules may be altered, and the synthesis
of DNA, RNA and protein may be temporarily inhibited immediately
after irradiation. New synthesis is evident by 24 hours and is
maximal by 60 hours. Harrison's Principles of Internal Medicine,
Petersdorf et al., eds., 10th Edition, McGraw-Hill Book Company,
New York, N.Y., p. 276 (1983). Thus, administration of the acyl
deoxyribonucleosides of the invention provides optimal levels of
deoxyribonucleosides during the period of DNA repair after sunlight
exposure to ensure maximal cellular protection and repair.
[0109] The acyl deoxyribonucleoside composition may be formulated
as part of a suntan lotion that may be applied before or after
exposure to sunlight. The suntan lotion may also comprise one or
more sun blockers such as PABA, esters of PABA, and other non-PABA
chemical sunscreens. See Harrison, supra, p. 279. The acyl
deoxynucleotides are absorbed by the skin and taken up by cells.
The acyl deoxyribonucleosides are then cleaved by tissue esterases
to give free deoxyribonucleosides in amounts effective for repair
of sunlight-induced chromosomal damage. The combination of the acyl
deoxyribonucleoside compositions and a sun blocker such as PABA
offers maximal protection of the skin from the sun.
[0110] The acyl deoxyribonucleoside compositions of the invention
also find use in ameliorating the effects of aging by providing a
high and sustained level of deoxyribonucleosides to enhance the
natural DNA repair processes of cells, and thereby, treating the
naturally occurring progressive accumulation of damage to DNA which
occurs on aging. Compositions for treatment or amelioration of the
effects of aging may be applied topically, in the form of a skin
lotion, or may be administered orally or parenterally.
[0111] There are conditions other than radiation damage in which
exogenous deoxyribonucleosides or derivatives thereof have useful
therapeutic applications. Deoxyribonucleic acid has been used to
accelerate wound cicatrization or healing, and also to accelerate
liver regeneration in experimental animals. It is likely that in
these situations, as well as in the situations where DNA is used to
promote survival after irradiation of animals, the DNA is serving
as a storage depot for deoxyribonucleosides, which gradually
releases the deoxyribonucleotides and deoxyribonucleosides during
enzymatic degradation.
[0112] Administration of acylated deoxyribonucleosides, as
described herein, is a method for delivering deoxyribonucleosides
to tissues which is preferable to the administration of foreign DNA
for the purpose of improving wound healing or tissue regeneration.
Unlike DNA, acylated deoxyribonucleosides are effective after oral
administration; they are also nonantigenic and are much easier to
purify than DNA.
[0113] The composition of the present invention may also be
administered to enhance the healing of damaged tissue. Such damaged
tissue includes skin wounds (e.g., punctures, lacerations,
abrasions, etc.), burned tissue (skin, etc.), diseased or damaged
liver (from surgery or other wounds of the liver, or from cirrhosis
or diabetes, etc.), damaged heart muscle (e.g., from myocardial
infarction), and damaged bone marrow (e.g., after radiation
treatment or chemotherapeutic treatment).
[0114] For the purpose of treating-skin wounds or burns, the
compositions may be applied topically as part of a skin lotion or
cream, or as part of a bioerodible polymer.
[0115] Preferred acyl substituent groups of 2'-deoxyadenosine,
2'-deoxyguanosine, 2'-deoxycytidine, and 2'-deoxythymidine are
those which form the acetate, valerate and palmitate esters.
[0116] Preferred deoxyadenosine derivatives comprise
N.sup.6-palmitoyl-3',5'-diacetyl-2'-deoxyadenosine,
N.sup.6-palmitoyl-5'-valeryl-2'-deoxyadenosine, and
5'-valeryl-2'-deoxyadenosine.
[0117] Preferred deoxyguanosine derivatives comprise
N.sup.2-palmitoyl-3',5'-diacetyl-2'-deoxyguanosine, and
5'-palmitoyl-2'-deoxyadenosine.
[0118] Preferred deoxycytidine derivatives comprise
N.sup.4-palmitoyl-2'-deoxycytidine, 5'-palmitoyl-2'-deoxycytidine,
N.sup.4-palmitoyl-3',5'-diacetyl-2'-deoxycytidine, and
3',5'-dipalmitoyl-2'-deoxycytidine.
[0119] Preferred deoxythymidine derivatives comprise
5'-palmitoyl-2-deoxythymidine and
N.sup.3-palmitoyl-3',5'-diacetyl-2-deox- ythymidine.
[0120] Compositions within the scope of the invention include those
which contain mixtures of the acyl derivatives of the
deoxyribonucleosides in amounts effective to achieve its intended
purpose. Such compositions may contain 0 to 50 mole percent of the
acyl derivative of deoxycytidine, 0 to 50 mole percent of the acyl
derivative of deoxyguanosine, 0 to 50 mole percent of the acyl
derivative of deoxythymidine and 0 to 50 mole percent of the acyl
derivative of deoxyadenosine, with the proviso that the total
content of the acyl deoxyribonucleosides adds up to 100 mole
percent.
[0121] A preferred composition contains 25 mole percent of the acyl
derivative of deoxycytidine, 25 mole percent of the acyl derivative
of deoxyguanosine, 25 mole percent of the acyl derivative of
deoxythymidine, and 25 mole percent of the acyl derivative of
deoxyadenosine.
[0122] For treatment of radiation-induced cellular damage, sunburn,
or to enhance wound healing, preferred dosages include amounts of
the acyl derivatives equivalent to 10 to 1000 mg of
2'-deoxyadenosine, 10 to 1000 mg of 2'-deoxyguanosine, 10 to 1000
mg of 2'-deoxycytidine and 10 to 1000 mg of 2'-deoxythymidine. For
example, the composition may comprise 13-1330 mg of
3',5'-diacetyl-2'-deoxyadenosine, 13-1310 mg of
3',3'-diacetyl-2'-deoxyguanosine, 14-1370 mg of
3',5'-diacetyl-2'-deoxycy- tidine and 14-1350 mg of
3',5'-diacetyl-2'-deoxythymidine. As is understood in the art, in
calculating such dosages, the equivalent amount of the
2'-deoxyribnucleoside alone is considered, i.e., the acyl
substituent and acid addition portion of any pharmaceutically
acceptable salt are not included in the calculation.
[0123] For a suntan lotion, 0.1 to 5% by weight of the above
compositions may be added. Generally, for this purpose, the acyl
derivative will be in the form of the free acyl
deoxyribonucleosides and not as the pharmaceutically acceptable
salts.
[0124] Methods of Preparation
[0125] When the acid source of the desired acyl derivative has
groups which interfere with the acylation reactions, e.g., hydroxyl
or amino groups, these groups may be blocked with protecting
groups, e.g., t-butyldimethylsilyl ethers or t-BOC groups,
respectively, before preparation of the anhydride. For example,
lactic acid may be converted to 2-t-butyldimethylsiloxypropionic
acid with t-butyldimethylchlorosilane- , followed by hydrolysis of
the resulting silyl ester with aqueous base. The anhydride may be
formed by reacting the protected acid with DCC. With amino acids,
the N-t-BOC derivative may be prepared, using standard techniques,
which is then converted to the anhydride with DCC. With acids
containing more than one carboxylate group (e.g., succinic,
fumaric, or adipic acid) the acid anhydride of the desired
dicarboxylic acid is reacted with a 2'-deoxyribonucleoside in
pyridine.
[0126] 3',5'-Diacyldeoxythymidine may be prepared according to
methods disclosed by Nishizawa et al., Biochem. Pharmacol. 14:1605
(1965), by treating deoxythymidine with 2.1 equivalents of an acid
anhydride of the desired acyl compound in pyridine followed by
heating to 80-85.degree. C. for at least one hour. Alternatively,
deoxythymidine may be treated with 2.1 equivalents of an acid
chloride in pyridine at room temperature (see Example 1).
[0127] The 5'-hydroxyl group of deoxythymidine may be selectively
acylated with 1 equivalent of the acid anhydride of the desired
acyl compound in pyridine, which is heated to 80-85.degree. C.,
according to Nishazawa, et al. Alternatively, the acid chloride (1
equivalent) may be reacted with deoxythymidine in pyridine and DMF
at room temperature according to Baker et al., J. Med. Chem.
21:1218 (1978). (See Example 2.)
[0128] The 3'-hydroxyl group of deoxythymidine may be selectively
acylated by selectively forming the 5'-O-t-butyldimethylsilyl
derivative with 1.2 equivalents of t-butyldimethylchlorosilane in
DMF containing imidizole, followed by acylation of the 3'-hydroxyl
group with the appropriate acid anhydride, and cleavage of the
5'-t-butyldimethyl silyl ether according to Baker et al. (See
Example 3.)
[0129] 3',5-Diacyldeoxycytidine may be prepared according to a
method adapted from Gish et al., J. Med. Chem. 14:1159 (1971), by
treating deoxycytidine hydrochloride with 2.1 equivalents of the
appropriate acid chloride in DMF. (See Example 5.)
[0130] The 5'-hydroxy group of deoxycytidine may be selectively
acylated by treating deoxycytidine hydrochloride with 1.1
equivalents of the appropriate acid anhydride in DMF. Gish et al.
(See Example 6.)
[0131] The N.sup.4-amino group of deoxycytidine may be selectively
acylated by treating deoxycytidine with 1.5 equivalents of the
appropriate acid anhydride in pyridine which may contain DMF.
Alternatively, deoxycytidine may be treated with 1.5 equivalents of
the desired acid anhydride in pyridine and DMF. Another procedure
for the selective acylation of the N.sup.4-amino group of
deoxycytidine involves treatment with an excess of the appropriate
acid anhydride in a mixture of water and a water-miscible solvent.
(See Example 7.)
[0132] 3',5',N.sup.4-Triacyl-2'-deoxycytidine, where all the acyl
groups are the same, may be prepared by treating 2'-deoxycytidine
with at least 3 equivalents of an acid chloride or anhydride
derived from the desired acyl group, in pyridine. To prepare the
triacyl derivatives of deoxycytidine where the N.sup.4-acyl group
is different from the 3',5'-acyl groups, the desired N.sup.4-amino
group may be first selectively acylated as described above,
followed by acylation of the 3' and 5'-hydroxy groups with the
desired acid anhydride. Alternatively, the 3',5'-diacyl derivative
may be prepared first, followed by acylation of the N.sup.4-amino
group. (See Example 8.)
[0133] The 3',5'-diacyl derivative of deoxyadenosine may be
prepared by treatment with 2.1 equivalents of the appropriate acid
chloride in DMF. (Adapted from Gish et al., see Example 9.)
[0134] The 5'-hydroxyl group of deoxyadenosine may be selectively
acylated by treatment of deoxyadenosine hydrochloride with 1.1
equivalents of the desired acid chloride in DMF. (Adapted from Gish
et al., see Example 10.)
[0135] The N.sup.6-amino group of deoxyadenosine may be selectively
acylated by treating deoxyadenosine with the desired acid chloride
(1.5 equivalents) in a mixture of pyridine and DMF. Alternatively,
deoxyadenosine may be treated with 1.5 equivalents of the desired
acid anhydride in pyridine and DMF (adapted from Sasaki et al.,
Chem. Pharm. Bull. 15:894 (1967)). The N.sup.6-amino group may also
be selectively acylated by treatment of deoxyadenosine with 2
equivalents of an appropriate acid anhydride in a mixture
comprising water and a water-miscible solvent (adapted from Akiyama
et al., Chem. Pharm. Bull. 26:981 (1978)) (See Example 11.)
[0136] The 3',5'-hydroxyl groups and the N.sup.6-amino group may
all be acylated by treatment of deoxyadenosine with at least 4
equivalents of the appropriate acid chloride or acid anhydride in
pyridine. (See Example 12.)
[0137] Alternatively, the N.sup.6-amino group of deoxyadenosine may
be first selectively acylated as described above, followed by
acylation of the 3' and 5' hydroxy groups. Another approach
comprises selective acylation of the 3' and 5'-hydroxy groups of
deoxyadenosine as described above, followed by acylation of the
N.sup.6-amino group. These methods give triacyl derivatives where
the N.sup.6-acyl group is different from the 3' and 5' acyl
groups.
[0138] 3,5-Diacyl-2'-deoxyguanosine may be prepared by treating
deoxyguanosine hydrochloride with 2.1 equivalents of the
appropriate acid chloride in DMF. (Adapted from Gish et al., see
Example 13.)
[0139] The 5'-hydroxyl group of deoxyguanosine may be selectively
acylated by treatment of deoxyguanosine hydrochloride with 1.1
equivalents of the appropriate acid chloride in DMF. (Adapted from
Gish et al., see Example 14.)
[0140] The N.sup.2-amine of the purine ring of deoxyguanosine may
be selectively acylated by treating deoxyguanosine with 1.5
equivalents of the appropriate acid anhydride in pyridine or a
mixture of pyridine and DMF (adapted from Sasaki et al., Chem.
Pharm. Bull. 13:894 (1967). Alternatively, deoxyguanosine may be
treated with a twofold excess of the appropriate acid anhydride in
a mixture of water and a water-miscible solvent (adapted from
Akiyama et al., see Example 15).
[0141] 3',5',N.sup.2-triacyldeoxyguanosine, where all the acyl
groups are the same, may be prepared by treating deoxyguanosine
with 4 equivalents of the appropriate acid chloride or acid
anhydride in pyridine. (See Example 16.)
[0142] To prepare the triacyl derivative of deoxyguanosine where
the N.sup.2-acyl group is different than the 3' and 5' acyl groups,
the N.sup.2-acyl group may be selectively acylated as described
above, followed by acylation of the 3' and 5' hydroxyl groups.
Alternatively, the 3' and 5' hydroxyl groups may be acylated first,
followed by acylation of the N.sup.2-amino group.
[0143] These acyl compositions may be administered chronically to
an animal which is at risk of either exposure to radiation,
sunlight or chemical mutagens. The acyl compositions of the
invention may also be administered after exposure to radiation,
sunlight or chemical mutagens or after a wound is inflicted to
enhance the repair of DNA and thereby to ameliorate the damage and
promote survival of the animal. Advantageously, the compositions of
the invention may be administered before or after radiotherapy or
chemotherapy to ameliorate undesired side effects of the
treatment.
[0144] The acyl compositions of the invention may also be
coadministered with other radioprotective compounds such as
WR-2721, NAC, DDC, cysteamine, 2 mercaptoethanol,
mercaptoethylamine, dithiothreitol, glutathione,
2-mercaptoethanesulfonic acid, WR-1065, nicotinamide,
5-hydroxytryptamine, 2- -aminoethyl-isothiouronium-Br-Hbr, glucans,
GLP/BO4, GLP/BO5, OK-432, Biostim, PSK, Lentinan, Schizophyllan,
Rhodexman, Levan, Mannozym, MVE-2, MNR, MMZ, IL-2, TNF, thymic
factor TF-5, glutathione peroxidase, superoxide dismutase,
catalase, glutathione reductase, glutathione transferase, selenium,
CdCl.sub.2, MrCl.sub.2, Zn acetate, vitamin A, beta carotene,
prostaglandins, tocopherol, and methylene blue. The administration
of these protective compounds along with the acyl derivatives of
the invention provides protection greater than if the acyl
derivatives or the other agents were given alone.
[0145] Mercaptoethylamine (cysteamine) may be prepared according to
Gabriel, L., Ber. 31:2837 (1898); Knorr, R., Ber. 36:1281 (1903);
Mills et al., J. Am. Chem. Soc. 62:1173 (1940), and Wenker et al.,
J. Am. Chem. Soc. 62:1173 (1940). AET may be prepared according to
Clinton et al., J. Am. Chem. Soc. 70:950 (1948); Funahashi, M., J.
Agric. Chem. Soc. Japan 27:775 (1953); Chemical Abstr. 49:157376
(1955) and Doherty et al., J. Am. Chem. Soc. 79:5667 (1957).
5-Hydroxytryptamine can be prepared according to Specter et al., J.
Am. Chem. Soc. 73:5514 (1951); Hamlin, U.S. Pat. No. 2,715,129
(1955); U.S. Pat. No. 2,947,757 (1960). For a review, see Erspamer
in E. Jucker, Progress in Drug Research 3:151-367 (1961). WR-2721
and WR-1065 may be obtained from the Walter Reed Army Medical
Center, Washington, D.C. For a synthesis of 2-mercaptoethanol, see
Woodward, J. Chem. Soc. 1892 (1948); Peppel et al., U.S. Pat. No.
2,401,665 (1964); U.S. Pat. No. 3,394,192 (1968). Dithiothreitol
may be prepared according to Evans et al., J. Chem. Soc. 253
(1949). Glutathione can be prepared according to du Vigneaud et
al., Biochem. Prepar. 2:87 (1952); Goldschmidt et al., Ber. 97:2434
(1964); and Ozawa et al., Bull. Chem. Soc. Japan 53:2592 (1980).
2-Mercaptoethanesulfonic acid sodium salt may be prepared according
to Schramm et al., J. Am. Chem. Soc. 77:6231 (1955); Reppe et al.,
Ann. 601:111 (1956); U.S. Pat. No. 3,567,835 (1971). Nicotinamide
may be prepared according to Truchan et al., U.S. Pat. No.
2,993,051 (1961); Gasson et al., U.S. Pat. No. 2,904,552 (1959).
Glucans may be isolated from Saccharomyces cerevisiae according to
Woods et al., Sci 142:178 (1978). OK-432 may be isolated from Saito
et al., Infec. Immun 26:779 (1979). PSK may be isolated from
Coriolus versicolor according to Otsuka, S., et al., Japanese
Patent No. 7,308,489 (1973). Chemical Abstr. 80:41025g (1974).
Lentinan may be prepared according to Abe et al., Gann 74:273
(1983). Schizophyllan may be prepared according to Saito et al.,
Infec. Immun 26:779 (1979). Rhodexman may be prepared according to
Elinov, Int. J. Immun. 4:265 (1982). Mannozym may be prepared
according to Nagy et al., Gen. Pharm. 12:A31 (1981). Polymannae
Rhoderamine may be extracted from Rhodotorula rubra according to
Komov, Vopr. Med. Khim. 21:351 (1975).
[0146] Cytokines IL-2 and TNF may be prepared according to Godard
et al., J. Immuno. Meth 70:233 (1984), and Brown, Photo Sci En.
22:22 (1978), respectively. Thymic peptides may be isolated
according to Tomazic et al., Proc. Am. Ass. Cancer Res. 24:196
(1983). Glutathione peroxidase may be isolated according to Aisaka
et al., Agro Biol. Chem. 47:1107 (1983). Superoxide dismutase can
be prepared according to Bio/Technology 5:363-365 (1987). Catalase
may be isolated according to Lolli, U.S. Pat. No. 2,703,779;
Schroeder et al., Biochim. Biohpys. Acta. 58:611 (1962); Dan, U.S.
Pat. No. 2,992,167 (1961); Faucett et al., U.S. Pat. No. 3,102,081
(1963). Glutathione reductase may be isolated according to
Muramatsu et al., Jap. Soc. Sci. Fisheries 46:757 (1980).
Glutathione transferase may be isolated according to Burgess et
al., Fed. Proc. 41:1738 (1982). Bis-(3,5-diisopropylsalicylato)
copper may be prepared according to Sorenson, J. Med. Chem. 27:1747
(1984). 3-Mercapto-2-hydroxypropyl ether of dextran may be prepared
according to Wieczorek, Arch. Immunol. Ther. Exp. (Warsz) 31:715
(1983). The immunostimulant IL-1 may be prepared according to
Krueger et al., Fed. Proc. 42:356 (1983). Vitamin A may be
extracted from fish liver oil according to Rarrer et al., Helv.
Chim. Acta. 16:625 (1933); Heilvon et al., Biochem. J. 26:1178,
1194 (1932). Beta carotene may be isolated from carrots according
to Willstatter et al., Z. Physiol. Chem. 64:47 (1910); Kuhn et al.,
Ber. 64:1349 (1931); Barnett et al., U.S. Pat. No. 2,848,508
(1958). For preparations of prostaglandins E.sub.1, E.sub.2,
F.sub.2 , I.sub.2 and X see The Merck Index, Tenth Edition,
Windholz, J., et al. (eds.), Merck and Col., Inc., Rahway, N.J.,
pp. 7777-7781 (1983). For a preparation of methylene blue see
Fierz-David, H. E., et al., Fundamental Processes of Dye Chemistry,
Interscience, New York, p. 311 (1949). N-arylacetyldehydroalanines
may be prepared according to Viehe, H. G., et al., Patent No.
WO/02523.
[0147] The pharmacologically active acyl derivatives may be
combined with suitable pharmaceutically acceptable carriers
comprising excipients and auxiliaries which facilitate processing
of the active compounds. These can be administered as tablets,
dragees, capsules, and suppositories. The compositions can be
administered orally, rectally, vaginally, or released through the
buccal pouch of the mouth, and may be applied in solution form by
injection, orally or by topical administration. The compositions
may contain about from 0.1 to 99%, preferably from about 50 to 90%
of the active compound(s), together with the excipient(s).
[0148] The pharmaceutical preparations of the present invention are
manufactured in a manner which is itself known, for example, by
means of conventional mixing, granulating, dragee-making,
dissolving, or lyophilizing processes. Thus, pharmaceutical
preparations for oral use can be obtained by combining the active
compound(s) with solid excipients, optionally grinding the
resulting mixture and processing the mixture of granules, after
adding suitable auxiliaries, if desired or necessary, to obtain
tablets or dragee cores.
[0149] Suitable excipients include fillers such as sugars, for
example lactose, sucrose, mannitol or sorbitol, cellulose
preparations and/or calcium phosphates, for example tricalcium
phosphate or calcium hydrogen phosphate, as well as binders such as
starch paste, using, for example, maize starch, wheat starch, rice
starch or potato starch, gelatin, tragacanth, methyl cellulose,
hydroxypropylmethyl cellulose, sodium carboxymethyl cellulose,
and/or polyvinyl pyrrolidone.
[0150] If desired, disintegrating agents may be added such as the
above-mentioned starches and also carboxymethylstarch, cross-linked
polyvinyl pyrrolidone, agar, or algenic acid or a salt thereof,
such as sodium alginate.
[0151] Auxiliaries are, above all, flow-regulating agents and
lubricants, for example, silica, talc, steric acid or salts
thereof, such as magnesium stearate or calcium stearate, and/or
polyethylene glycol. Dragee cores are provided with suitable
coatings which, if desired, are resistant to gastric juices. For
this purpose, concentrated sugar solutions may be used, which may
optionally contain gum arabic, talc, polyvinyl pyrrolidone,
polyethylene glycol and/or titanium dioxide, lacquer solutions and
suitable organic solvents or solvent mixtures. In order to produce
coatings resistant to gastric juices, solutions of suitable
cellulose preparations such as acetylcellulose phthalate or
hydroxypropylmethylcellulose phthalate are used. Dye stuffs or
pigments may be added to the tablets or dragee coatings, for
example, for identification or in order to characterize different
combinations of compound doses.
[0152] Other pharmaceutical preparations 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 contain the active compounds in the
form of granules which may be mixed with fillers 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 are preferably dissolved or suspended in
suitable liquids such as fatty oils, liquid paraffin, or
polyethylene glycols. In addition, stabilizers may be added.
[0153] Possible pharmaceutical preparations which can be used
rectally include, for example, suppositories which consist of a
combination of active compounds with a suppository base. Suitable
suppository bases are, for example, natural or synthetic
triglycerides, paraffin hydrocarbons, polyethylene glycols or
higher alkanols. In addition, it is also possible to use gelatin
rectal capsules which consist of a combination of the active
compounds with a base. Possible base materials include, for
example, liquid triglycerides, polyethylene glycols, or paraffin
hydrocarbons.
[0154] Suitable formulations for parenteral administration include
aqueous solutions of the active compounds in water soluble form,
for example, water soluble salts. In addition, suspensions of the
active compounds as appropriate oily injection suspensions may be
administered. Suitable lipophilic solvents or vehicles include
fatty oils, for example, sesame oil, or synthetic fatty acid
esters, for example, ethyl oleate or triglycerides. Aqueous
injection suspensions may include substances which increase the
viscosity of the suspension which include, for example, sodium
carboxymethylcellulose, sorbitol, and/or dextran. Optionally, the
suspension may also contain stabilizers.
[0155] The acyl deoxyribonucleosides may be formulated as part of a
skin lotion or suntan lotion for topical administration. Suitable
formulations for topical administration include appropriate oily
suspensions or solutions. Suitable lipophilic solvents or vehicles
include fatty oils, for example sesame oil or coconut oil, or
synthetic fatty acid esters, for example ethyl oleate or
triglycerides. These topical formulations may be used to treat
damaged tissue such as skin wounds or burns, or to treat or prevent
sunlight induced cellular damage (sunburn).
[0156] For purposes of enhancing wound healing, the compositions of
the present invention may be formulated as part of bioerodible
microcapsules for topical administration. Such microcapsules may
comprise, for example, polylactate or lactate-glycolate copolymers.
See Weise, D. L. et al., Drug Carriers in Biology and Medicine,
Gregoriadis, G. et al., Academic Press, NY p. 237-270 (1979).
[0157] The following examples are illustrative, but not limiting of
the methods and compositions of the present invention. Other
suitable modifications and adaptations of a variety of conditions
and parameters normally encountered in clinical therapy which are
obvious to the those skilled in the art are within the spirit and
scope of this invention.
EXAMPLES OF METHODS TO PREPARE COMPOUNDS OF THE INVENTION
Example 1
Preparation of 3',5'-Diacyl-2'-deoxythymidine from acid
anhydrides
[0158] 2'-Deoxythymidine is dissolved in anhydrous pyridine at room
temperature. 2.1 molar equivalents of the acid anhydride of the
desired acyl compound (e.g., acetic anhydride, lactate anhydride,
butyric anhydride, etc.) is then added. The reaction mixture is
then heated to 80-85.degree. C. for 1 to 4 hours, cooled, poured
into ice water, and the esters recovered by extraction with
chloroform or a similar solvent. The chloroform is then washed with
ice-cold 0.01 N sulfuric acid, 1% aqueous sodium bicarbonate, and
finally water. After drying with sodium sulfate, the chloroform is
evaporated and the residual oil or crystals are subjected to
chromatography (adapted from Nishizawa et al., Biochem. Pharmacol.
14:1605 (1965)).
[0159] From Acid Chlorides:
[0160] To 2'-deoxythymidine dissolved in anhydrous pyridine is
added, at 5.degree. C., 2.1 molar equivalents of the acid chloride
of the desired acyl compound (e.g., palmitoyl chloride, acetyl
chloride, etc.). The mixture is held at room temperature overnight,
added to ice water, and worked up as indicated above (adapted from
Nishizawa).
Example 2
Preparation of 5'-Acyl-2'-deoxythymidine
[0161] To 2'-deoxythymidine dissolved in anhydrous pyridine is
added, at room temperature, 1.0 molar equivalent of the acid
anhydride of the desired acyl compound. The reaction is then heated
to approximately 80-85.degree. C. for several hours, cooled, poured
into ice water, and the esters recovered by extraction with
chloroform or a similar solvent. The chloroform is then washed in
ice-cold 0.01 N sulfuric acid, 1% aqueous sodium bicarbonate, and
finally water. After drying with sodium sulfate, the chloroform is
evaporated and the residual oil or crystals are subjected to
chromatography. The major product, which is isolated by
chromatography is the 5' substituted ester (adapted from Nishizawa
et al.
[0162] Alternatively, selectively 5' acylation of deoxythymidine
may be accomplished by suspending 2'-deoxythymidine in a mixture of
pyridine and N,N-dimethylformamide cooled to 0.degree. C. in an ice
bath. 1.0 molar equivalent of the acid chloride of the desired acyl
compound is added dropwise to the mixture, which is stirred at
9.degree. C. for 12-24 hours. Water is then added to stop the
reaction, and then the solvents are evaporated in vacuo at
50.degree. C. The residue is dissolved in methanol and purified by
chromatography on silica gel (adapted from Baker et al., J. Med.
Chem. 21:1218 (1978).
Example 3
Preparation of 3'-Acyl-2'-deoxythymidine
[0163] To a stirred suspension of 2'-deoxythymidine in dry
N,N-dimethylformamide is added 2.4 molar equivalents of imidazole
followed by 1.2 molar equivalents of t-butyldimethylchlorosilane.
The mixture is stirred with protection from moisture at room
temperature for 20 hours, at which time the solvent is removed at
50.degree. C. in vacuo. The residue is dissolved in 15 ml of ethyl
acetate, washed, and evaporated to give a syrup from which is
obtained, by crystallization from hot chloroform by the addition of
hexane to the point of opalescence,
5'-(t-butyldimethylsilyl)-2'-deoxythymidine.
[0164] To a stirred suspension of
5'-(t-butylmethylsilyl)-2'-deoxythymidin- e in dry pyridine cooled
to 0.degree. C. is added 1.1 molar equivalents of the appropriate
acid anhydride of the desired acyl compound, and the mixture is
stirred with protection from moisture for 20 hours at 0-5.degree.
C., at which time the reaction is terminated by addition of a few
ml of water. The solvent is evaporated and the residue is extracted
and evaporated to give a thick, clear syrup, which is then dried in
vacuo at 25.degree. C.
[0165] The t-butylmethylsilyl group is removed with glacial acetic
acid and tetrabutylammonium fluoride in tetrahydrofuran, yielding
the desired 3'-acyl-2'-deoxythymidine derivative (adapted from
Baker et al.
Example 4
Preparation of N.sup.3-Acyl-2'-deoxythymidine
[0166] The acylation of the secondary amine in the 3 position of
the pyrimidine ring is accomplished by reacting
3',5'-diacyldeoxythymidine with 1.1 molar equivalents of the acid
chloride of the desired acyl substituent in an aprotic solvent
(such as ether, dioxane, chloroform, ethyl acetate, acetonitrile,
pyridine, dimethylformamide, and the like) in the presence of 1-5
molar equivalents of an organic base (especially aromatic amines
such as pyridine, trialkylamines, or N,N-dialkylanilines) (adapted
from Fuji et al., U.S. Pat. No. 4,425,335). The acyl substituent on
the secondary amine can be the same or different from those on the
hydroxyl groups of the ribose moiety.
Example 5
Preparation of 3',5'-Diacyl-2'-deoxycytidine
[0167] 2-Deoxycytidine hydrochloride is dissolved in
N,N-dimethylformamide. 2.1 molar equivalents of the acid chloride
of the desired acyl substituent is added, and the mixture is
stirred overnight at room temperature. The reaction mixture is
concentrated in vacuo to an oil, and triturated with a mixture of
ethyl acetate and diethyl ether or similar solvents. The oil is
then triturated with 1N sodium hydrogen carbonate. The crystalline
solid is collected, washed with water, dried, and recrystallized
(adapted from Gish et al., J. Med. Chem. 14:1159 (1971)).
Example 6
Preparation of 5'-Acyl-2'-deoxycytidine
[0168] 2-Deoxycytidine hydrochloride is dissolved in
N,N-dimethylformamide. 1.1 molar equivalents of the acid chloride
of the desired acyl substituent is added, and the mixture is
stirred overnight at room temperature. The reaction mixture is
concentrated in vacuo to an oil, and triturated with a mixture of
ethyl acetate and diethyl ether or similar solvents. The oil is
then triturated with 1N sodium hydrogen carbonate. The crystalline
solid is collected, washed with water, dried, and recrystallized
(adapted from Gish et al.).
Example 7
Preparation of N.sup.4-Acyl-2'-deoxycytidine
[0169] The N.sup.4-amino group of 2'-deoxycytidine is the best
nucleophile among the amino and hydroxyl functionalities of
deoxycytidine. Selective N.sup.4-acylation can be accomplished by
treating 2'-deoxycytidine with appropriate acid anhydrides in
pyridine or a mixture of pyridine and N,N-dimethylformamide.
Specifically, deoxycytidine is suspended in pyridine, 1.5 molar
equivalents of desired acid anhydride is added, and the mixture is
refluxed for about 2 hours. The solvent is removed in vacuo, and
the resulting white solid is recrystallized.
[0170] Alternatively, 2'-deoxycytidine is dissolved in 70:30
pyridine:n,N-dimethylformamide. 1.5 molar equivalents of the acid
anhydride of the desired acyl substituent is added, and the mixture
is stirred overnight at room temperature, after which it is poured
into water and stirred. The solvent is removed in vacuo to leave a
white solid, which is recrystallized (adapted from Sasaki et al.,
Chem. Pharm. Bul. 15:894 (1967)).
[0171] An alternative procedure is to dissolve 2'-deoxycytidine in
a mixture of water and a water-miscible organic solvent (such as
dioxane, acetone, acetonitrile, N,N-dimethylformamide,
dimethylsulfoxide, tetrahydrofuran, etc.) and to treat that
solution with about a twofold excess of an appropriate acid
anhydride. For example, 1 gram of 2'-deoxycytidine, dissolved in 5
ml of water, is mixed with 15 to 100 ml dioxane (more dioxane is
needed for more lipophilic substituents), and 2 molar equivalents
of the acid anhydride of the desired acyl substituent is added. The
mixture is stirred for 5 hours at 80.degree. C. (or for 48 hours at
room temperature), and then the solvent is removed in vacuo. The
residue is washed with hexane or benzene, and recrystallized
(adapted from Akiyama et al., Chem. Pharm. Bull. 26:981
(1978)).
Example 8
Preparation of 3',5'N.sup.4-Triacyl-2'-deoxycytidine
[0172] Compounds in which the acyl substituent of the N.sup.4 amino
group and the hydroxyl groups of the ribose ring of deoxycytidine
are the same (e.g., triacetyl deoxycytidine) are prepared by
dissolving or suspending 2'-deoxycytidine in dry pyridine, adding
at least 3 molar equivalents of the acid chloride or acid anhydride
of the desired substituent, and stirring the mixture overnight at
room temperature. The solvent is removed in vacuo and the residue
is washed and recrystallized.
Example 9
Preparation of 3',5'-Diacyl-2'-deoxyadenosine
[0173] 2'-Deoxyadenosine hydrochloride is dissolved in
N,N-dimethylformamide. 2.1 molar equivalents of the acid chloride
of the desired acyl substituent is added, and the mixture is
stirred overnight at room temperature. The reaction mixture is
concentrated in vacuo to an oil, and triturated with a mixture of
ethyl acetate and diethyl ether or similar solvents. The oil is
then triturated with 1N sodium hydrogen carbonate. The crystalline
solid is collected, washed with water, dried, and recrystallized
(adapted from Gish et al.).
Example 10
Preparation of 5'-Acyl-2'-deoxyadenosine
[0174] 2'-Deoxyadenosine hydrochloride is dissolved in
N,N-dimethylformamide. 1.1 molar equivalents of the acid chloride
of the desired acyl substituent is added, and the mixture is
stirred overnight at room temperature. The reaction mixture is
concentrated in vacuo to an oil, and triturated with a mixture of
ethyl acetate and diethyl ether or similar solvents. The oil is
then triturated with 1N sodium hydrogen carbonate. The crystalline
solid is collected, washed with water, dried, and recrystallized
(adapted from Gish et al.
Example 11
Preparation of N.sup.6-Acyl-2'-deoxyadenosine
[0175] The N.sup.6-amino group of deoxyadenosine is the best
nucleophile among the amino and hydroxyl functionalities of
deoxyadenosine. Selective N.sup.6-acylation can be accomplished by
treatnig 2'-deoxyadenosine with appropriate acid anhydrides in
pyridine or a mixture of pyridine and N,N-dimethylformamide.
Specifically, deoxyadenosine is suspended in pyridine, 1.5 molar
equivalents of desired acid anhydride is added, and the mixture is
refluxed for about 2 hours. The solvent is removed in vacuo, and
the resulting white solid is recrystallized.
[0176] Alternatively, 2'-deoxyadenosine is dissolved in 70:30
pyridine:n,N-dimethylformamide. 1.5 molar equivalents of the acid
anhydride of the desired acyl substituent is added, and the mixture
is stirred overnight at room temperature, after which it is poured
into water and stirred. The solvent is removed in vacuo to leave a
white solid, which is recrystallized (adapted from Sasaki et al.,
Chem. Pharm. Bul. 15:894 (1967)).
[0177] An alternative procedure is to dissolve 2'-deoxyadenosine in
a mixture of water and a water-miscible organic solvent (such as
dioxane, acetone, acetonitrile, N,N-dimethylformamide,
dimethylsulfoxide, tetrahydrofuran, etc.) and to treat that
solution with about a twofold excess of an appropriate acid
anhydride. For example, 1 gram of 2'-deoxyadenosine, dissolved in 5
ml of water, is mixed with 15 to 100 ml dioxane (more dioxane is
needed for more lipophilic substituents), and 2 molar equivalents
of the acid anhydride of the desired acyl substituent is added. The
mixture is stirred for 5 hours at 80.degree. C. (or for 48 hours at
room temperature), and then the solvent is removed in vacuo. The
residue is washed with hexane or benzene, and recrystallized
(adapted from Akiyama et al.).
Example 12
Preparation of 3',5'N.sup.6-Triacyl-2'-deoxyadenosine
[0178] Compounds in which the acyl substituent of the N.sup.6 amino
group and the hydroxyl group of the ribose ring of deoxyadenosine
are the same (e.g., tretraacetyl deoxyadenosine) are prepared by
dissolving or suspending 2'-deoxyadenosine in dry pyridine, adding
at least 4 molar equivalents of the acid chloride or acid anhydride
of the desired substituent, and stirring the mixture overnight at
room temperature. The solvent is removed in vacuo and their residue
is washed and recrystallized.
Example 13
Preparation of 3',5'-Diacyl-2'-deoxyguanosine
[0179] 2'-Deoxyguanosine hydrochloride is dissolved in
N,N-dimethylformamide. 2.1 molar equivalents of the acid chloride
of the desired acyl substituent is added, and the mixture is
stirred overnight at room temperature. The reaction mixture is
concentrated in vacuo to an oil, and triturated with a mixture of
ethyl acetate and diethyl ether or similar solvents. The oil is
then triturated with 1N sodium hydrogen carbonate. The crystalline
solid is collected, washed with water, dried, and recrystallized
(adapted from Gish et al.).
Example 14
Preparation of 5'-Acyl-2'-deoxyguanosine
[0180] 2'-Deoxyguanosine hydrochloride is dissolved in
N,N-dimethylformamide. 1.1 molar equivalents of the acid chloride
of the desired acyl substituent is added, and the mixture is
stirred overnight at room temperature. The reaction mixture is
concentrated in vacuo to an oil, and triturated with a mixture of
ethyl acetate and diethyl ether or similar solvents. The oil is
then triturated with 1N sodium hydrogen carbonate. The crystalline
solid is collected, washed with water, dried, and recrystallized
(adapted from Gish et al.).
Example 15
Preparation of N.sup.2-Acyl-2'-deoxyguanosine
[0181] The N.sup.2-amino group of deoxyguanosine is the best
nucleophile among the amino and hydroxyl functionalities of
deoxyguanosine. Selective N.sup.2-acylation can be accomplished by
treatnig 2'-deoxyguanosine with appropriate acid anhydrides in
pyridine or a mixture of pyridine and N,N-dimethylformamide.
Specifically, deoxyguanosine is suspended in pyridine, 1.5 molar
equivalents of desired acid anhydride is added, and the mixture is
refluxed for about 2 hours. The solvent is removed in vacuo, and
the resulting white solid is recrystallized.
[0182] Alternatively, 2'-deoxyguanosine is dissolved in 70:30
pyridine:n,N-dimethylformamide. 1.5 molar equivalents of the acid
anhydride of the desired acyl substituent is added, and the mixture
is stirred overnight at room temperature, after which it is poured
into water and stirred. The solvent is removed in vacuo to leave a
white solid, which is recrystallized (adapted from Sasaki et
al.).
[0183] An alternative procedure is to dissolve 2'-deoxyadenosine in
a mixture of water and a water-miscible organic solvent (such as
dioxane, acetone, acetonitrile, N,N-dimethylformamide,
dimethylsulfoxide, tetrahydrofuran, etc.) and to treat that
solution with about a twofold excess of an appropriate acid
anhydride. For example, 1 gram of 2'-deoxyadenosine, dissolved in 5
ml of water, is mixed with 15 to 100 ml dioxane (more dioxane is
needed for more lipophilic substituents), and 2 molar equivalents
of the acid anhydride of the desired acyl substituent is added. The
mixture is stirred for 5 hours at 80.degree. C. (or for 48 hours at
room temperature), and then the solvent is removed in vacuo. The
residue is washed with hexane or benzene, and recrystallized
(adapted from Akiyama et al.).
Example 16
Preparation of 3',5'N.sup.2-Triacyl-2'-deoxyguanosine
[0184] Compounds in which the acyl substituent of the N.sup.2 amino
group and the hydroxyl groups of the ribose ring of deoxyguanosine
are the same (e.g., tretraacetyl deoxyguanosine) are prepared by
dissolving or suspending 2'-deoxyguanosine in dry pyridine, adding
at least 4 molar equivalents of the acid chloride or acid anhydride
of the desired substituent, and stirring the mixture overnight at
room temperature. The solvent is removed in vacuo and their residue
is washed and recrystallized.
Examples of Clinical Administration
[0185] Radiation Exposure
[0186] Three situations wherein acyl derivatives of
deoxyribonucleosides may be clinically useful in treating radiation
damage are 1) accidental exposure to ionizing radiation, as in a
nuclear accident; 2) exposure to X-radiation during radiography;
and 3) radiotherapy of cancer.
[0187] In the first case, acyl deoxyribonucleoside derivatives
should be administered in a formulation suitable for parenteral
injection, followed by oral administration several times per day of
doses equivalent to 0.5 to 2 grams of each of the four major
deoxyribonucleosides. It is essential that the derivatives of all
of the nucleosides be coadministered.
[0188] In the second case, X-ray exposure during diagnostic
radiography, acyl deoxyribonucleside derivatives are given orally
before and after exposure.
[0189] In the third case, during cancer radiotherapy, the acyl
ribonucleoside derivatives are particularly useful in restoring
bone marrow function after its undesirable but unavoidable
suppression during irradiation. Moreover, in formulations designed
to selectively deliver nucleosides to normal but not neoplastic
tissues, the acyl nucleoside derivatives will improve the
therapeutic index (ratio of efficacy to toxicity) of the radiation
treatment.
[0190] Wound Healing
[0191] In promoting the healing of skin wounds (whether surgical
incisions or accidental wounds), it is best to apply acyl
deoxyribonucleoside derivatives topically, either in an ointment or
in bioerodible microcapsules. A topical antibiotic might be
coadministered. The molar equivalent of 2 to 20 mg of a mixture of
all four major deoxyribonucleosides should be applied per square cm
of wound area, or 1 to 10 mg per cm of linear incision. The onset
of the earliest phases of wound healing in particular is
accelerated.
[0192] Liver Regneration
[0193] Acyl derivatives of deoxyribonucleosides are useful in
promoting regeneration of damaged or diseased liver, particularly
for accelerating regrowth after surgical removal of a portion of
the liver. In this case, oral administration of the derivatives is
preferable, in doses corresponding to the molar equivalents of 0.2
to 2 grams of each nucleoside. It is important that derivatives of
all four major deoxyribonucleosides be coadministered.
* * * * *