A method of improving DNA repair and reducing DNA damage and for reducing mutation frequency in skin for the purpose of reducing consequences of exposure to solar or ultraviolet radiation is disclosed. The methods comprise administering to the skin a composition containing deoxyribonucleosides in concentrations sufficient to enhance DNA repair or reduce mutation frequency in a vehicle capable of delivering effective amounts of deoxyribonucleosides to the necessary skin cells.

This invention relates generally to treatment and prevention of photodamage, genetic damage, and tumorigenesis in skin and other tissues caused by exposure to solar or ultraviolet radiation or other mutagens, comprising administration of deoxyribonucleosides or esters of deoxyribonucleosides to a mammal such as a human. These compounds are capable of reducing DNA damage, mutation frequency, and tumorigenesis when applied topically before, during, or after exposure to mutagenic radiation or chemical mutagens.
Exposure of skin to ultraviolet (or ionizing) radiation damages DNA, which if unrepaired or improperly repaired, can lead to carcinogenesis as well as contribute to acceleration of the aging process. DNA damage and consequent genomic instability are defining characteristics of both carcinogenesis and biological aging. Patients with defective DNA repair capabilities in diseases like xeroderma pigmentosa display premature skin aging and a very high incidence of skin cancers (Robbins and Moshell, J. Inv. Dermatol., 73:102-107, 1979) on sun-exposed areas of the skin. Pharmacological intervention in damage to skin due to solar or ultraviolet radiation has heretofore been largely restricted to agents like sunscreens or free-radical scavengers intended to prevent damage, or agents like retinoic acid or glycolic acid which are intended to remodel the surface of radiation-damaged skin without necessarily addressing the most fundamental mechanisms of cell or tissue damage and repair at the level of genomic integrity.
In practice, preventive measures like sunscreen use are not completely effective, and exposure to sunlight is not always anticipated. The incidence of skin cancers in the United States approaches 1,000,000 cases per year. Therefore, there exists a need for a therapeutic agent which will reduce the risk of development of skin cancer or other consequences of skin photodamage even when applied after exposure to sunlight has already occurred. Sunscreens and agents which induce or improve tanning are not useful in such situations, since they are only useful if applied prior to exposure to UV radiation. Moreover, there are situations wherein sunscreens and even endogenous melanin can actually enhance UV-induced DNA damage through photodynamic sensitization.
There have been several attempts to improve or accelerate DNA repair and to reduce the consequences of DNA damage in skin cells after damage has already occurred. The first major step in DNA repair is detection and excision of damaged portions of DNA. The viral enzyme T4 endonuclease V can accomplish this step with some forms of DNA damage. T4 endonuclease V, when packaged in epidermis-penetrating liposomes, has been shown to accelerate the rate of excision of pyrimidine dimers, the most common form of photolesion, in photodamaged skin of mice in vivo (Yarosh et al., Cancer Res. 52:4227-31, 1992). A bacterial extract has been reported to increase the rate of unscheduled DNA synthesis, which is often used as an index of DNA repair activity (Kludas and Heise, U.S. Pat. No. 4,464,362), in UV-exposed skin; however, this effect was not confirmed in a subsequent study (Natarajan et al., Mutation Research 206:47-54, 1988).
The key issue in DNA repair, however, is not necessarily the rate of lesion excision, but the fidelity of repair. Agents which accelerate the excision step of DNA repair can actually exacerbate damage if the cells are incapable of accurate repair synthesis at a rate that matches the rate of excision of damaged segments of DNA (Collins and Johnson, J. Cell Physiol. 99:125-137, 1979).
Deoxyribonucleosides or deoxyribonucleotides have been added to cells in culture with variable or divergent effects on DNA damage or mutagenesis in response to irradiation of the cells. In some cell types, e.g. lymphocytes, which have limited capabilities for de novo deoxyribonucleotide synthesis, exogenous deoxynucleosides are reported to improve cell survival after exposure to UV radiation (Yew and Johnson, Biochim. Biophys. Acta, 562:240-251, 1979; Green et al., Mutation Research, 350:239-246, 1996) or ionizing radiation (Petrovic et al., Int. J. Radiat. Biol., 18:243, 1970); no significant improvement in survival was seen after addition of deoxyribonucleosides to UV-irradiated normal human fibroblasts (Green et al., Mutation Research, 350:239-246, 1996). A crucial point is that increasing cell survival after genomic damage caused by UV radiation or other mutagens is not necessarily desirable. The process of programmed cell death, or apoptosis, is integrated with cellular mechanisms for detecting DNA damage. Thus, genomic damage which by itself is not sufficient to cause cell death, can trigger apoptosis, an active cellular suicide process, so that the DNA damage in the cell is not perpetuated in subsequent cell generations, with tumorigenesis as a possible outcome as genomic damage accumulates. The mechanisms for detecting genomic damage and inducing apoptosis involve cell-cycle regulating proteins such as the tumor-suppressor protein p53. Therefore, agents which promote cell survival (e.g. by inhibiting apoptosis) after irradiation are not necessarily anticarcinogenic, and may actually enhance mutation frequency and risk of malignant transformation by permitting survival of damaged cells that would otherwise be eliminated by apoptosis. A significant illustration of this principle is the demonstration that embryonic p53 knockout mice exposed to ionizing radiation in utero have a higher survival rate (live birth) than wild-type controls, but also have a much higher frequency of congenital defects (Norimura et al., Nature Medicine, 2:577-580).
In studies where the effect of exogenous deoxyribonucleosides on mutation frequency in UV-irradiated cells has been explicitly studied, variable results have been obtained. Bianchi and Celotti (Mutation Research 146:277-284, 1985) reported that thymidine or deoxycytidine at high concentrations increased the mutation frequency in UV-irradiated V79 Chinese hamster cells; no reduction in mutation frequency was observed at any concentrations of added nucleosides. Musk et al. (Mutation Research 227:25-30, 1989) reported that a mixture of deoxyribonucleosides which included excess deoxycytidine relative to the other nucleosides, reduced the mutation frequency in response to UV-C (254 nm) irradiation in MM96L melanoma cells, a cell line with a known constitutive excess of purine deoxyribonucleotides. In the same study, exogenous deoxyribonucleosides had no effect on mutation frequency in another neoplastic cell line, human HeLa cells, after exposure to UV-C radiation. It is important to note that UV-C radiation is not a component of solar radiation at the surface of the earth, since it is blocked effectively by the atmosphere (Pathak, 1974, in Sunlight and Man, ed. by T. B. Fitzpatrick, University of Tokyo Press, Tokyo, Japan, p. 815). The effect of deoxyribonucleosides on mutation frequency in cells exposed to solar radiation or UV radiation at wavelengths that are present in solar radiation was not tested, and the authors explicitly conclude their discussion with the statement xe2x80x9c . . . the lower [mutation] frequency in sun-[irradiated] compared with UVC-irradiated MM96L cells suggests that sunlight either does not perturb the deoxynucleoside pools or it induces a cellular response that is insensitive to nucleoside levels.xe2x80x9d
In addition to agents which inhibit apoptosis or improve DNA repair sufficiently to permit cell survival but not necessarily for correction of potentially tumorigenic mutations, growth factors in general (including those that are involved in normal wound healing responses like TGF-xcex2 or PDGF) act as tumor promoters.
U.S. Pat. No. 5,246,708 discloses the methods and compositions involving the use of mixtures of deoxyribonucleosides for promotion of the healing of wounds, ulcers, and burns, including those caused by ultraviolet or solar radiation.
Acyl derivatives of deoxyribonucleosides have been taught as delivery molecules for promoting entry of deoxyribonucleosides into the skin, as disclosed in U.S. patent application Ser. No. 08/466,379, filed Jun. 6, 1995, now U.S. Pat. No. 6,297,222. It is disclosed that acyl derivatives of deoxyribonucleosides can improve cellular repair and cell survival after damage to skin caused by radiation.
Oligodeoxyribonucleotides have been proposed as melanogenic stimuli, based on the idea that DNA damage, or excision products of DNA damage, might be cellular signals for increasing melanin production in the skin to help protect against subsequent damage. Gilchrest et al. (U.S. Pat. No. 5,470,577; WO Application Ser. No. 95/01773) proposed that exogenous DNA photodamage products may stimulate melanogenesis without actual damage to cellular DNA as a necessary intermediate step. The stated intention was to mimic the presence of cyclobutane pyrimidine dimers or other DNA photodamage products in order to provide the cell with false DNA damage signals that might trigger induction of melanogenesis in the absence of actual DNA damage. Treatment of melanoma cells in vitro and guinea pig skin in vivo with thymidine dinucleotide resulted in increases in melanin production. The authors stated that they believed that DNA fragments entered the cells, and even their nuclei, intact. They proposed that sunless tanning accomplished over a period of weeks by topical administration of oligodeoxyribonucleotides, especially thymidine dinucleotide, could protect skin by inducing melanin synthesis, with consequent reduction of passage of UV radiation into and through the skin.
Wiskemann (1974; in Sunlight and Man, ed. by T. B. Fizpatrick, University of Tokyo Press, Tokyo, Japan, p. 51) reported that systemic (intraperitoneal) administration the deoxyribonucleoside thymidine or the ribonucleosides and congeners adenosine, cyclic-AMP, uridine, cytidine increased the period of latency for extravasation of systemically administered Evan""s Blue dye in the skin in the first few hours after UV exposure, indicating a reduction in acute UV-induced edema. In this system, DNA administered after irradiation had no effect on extravasation of dye. The author also explicitly states that nucleobases incorporated into ointments do not penetrate the horny layer (the stratum corneum, the outer layer of enucleated keratinocytes comprising the main moisture barrier of skin) of human epidermis.
It is an object of the invention to provide compositions and methods for reducing mutation frequency, photaging, and tumorigenesis in skin, thereby attenuating consequences of exposure to solar and ultraviolet radiation and to other mutagens including endogenous oxidants.
It is an object of the invention to provide a composition that enhances DNA repair and prevents consequences of mutagenic radiation even when administered after damage or exposure to radiation or other mutagens has already occurred.
It is a primary object of this invention to provide compositions and methods for effectively preventing or treating consequences of exposure of the skin to solar and ultraviolet radiation and other environmental mutagens.
It is a further object of the invention to provide compositions and methods for improving the activity of chemical sunscreens.
It is a further object of the invention to provide compositions and methods for reducing deleterious effects of sunscreens and other compounds, exogenous and endogenous, which act as photosensitizing or photodynamic enhancers of UV-induced damage to skin.
It is a further object of the invention to provide compositions and methods for reducing some consequences of inflammatory skin and mucosal conditions, including psoriasis, dermatitis and inflammatory bowel disease.
The subject invention involves methods and compositions for improving DNA repair (or genomic fidelity) and reducing photodamage in skin exposed to ultraviolet radiation, ionizing radiation, or chemical mutagens by topical administration of compositions containing effective amounts of a source of deoxyribonucleosides. The compositions are capable of delivering deoxyribonucleosides to the necessary target cells. Such compositions are applied to the skin before, during or after exposure to solar, ultraviolet, or ionizing radiation, or chemical mutagens including but not limited to endogenous or exogenous sources of free radicals or nitric oxide. Treatment with compositions of the invention improves the net fidelity of DNA repair and thereby reduces mutation frequency and the risk of tumorigenesis in response to solar or ultraviolet radiation or other mutagens.
The invention provides methods and compositions for delivering deoxyribonucleosides to skin cells in concentrations sufficient to support and improve repair of damaged DNA and to reduce deleterious consequences of exposure of skin to radiation or chemical mutagens.
The deoxyribonucleosides are administered either as free deoxyribonucleosides, or as derivatives thereof which are converted to deoxyribonucleosides after application to the skin. Such derivatives include deoxyribonucleotides, oligonucleotides, DNA itself, and acyl derivatives of deoxyribonucleosides or other derivatives of deoxyribonucleosides which are converted to free deoxyribonucleosides by endogenous enzymes.
Methods and compositions of the invention also improve activity and reduce side effects of other agents used on skin for prophylactic, therapeutic, or cosmetic purposes, including but not limited to sunscreens, retinoids, alpha-hydroxy acids, methylxanthines, and DNA repair enzymes.
The invention also relates to compositions and methods for reducing deleterious consequences (e.g. cellular damage, especially to DNA, which can result in increased likelihood of mutations or other potentially carcinogenic damage to the genome) of endogenous and exogenous photochemically-active compounds or chromophores which act as photosensitizers or photodynamic enhancers of DNA damage caused by solar or ultraviolet radiation.
The invention, as well as other objects, features and advantages thereof will be understood more clearly and fully from the following detailed description, when read with reference to the accompanying results of the experiments discussed in the examples below.
DNA damage and repair is involved in the development of skin cancer and photoaging. The subject invention provides compounds which successfully improve the net fidelity of DNA repair. The subject invention will have important consequences in health care and will improve the cosmetic appearance of skin.
A. Definitions
The term xe2x80x9cdeoxyribonucleosidexe2x80x9d refers to any one of the four principle nucleoside constituents of DNA: deoxyadenosine, deoxycytidine, deoxyguanosine, and thymidine. The term xe2x80x9cribonucleosidexe2x80x9d refers to any one of the four major nucleoside constituents of RNA: adenosine, cytidine, guanosine, and uridine.
The term xe2x80x9cacyl derivative of a deoxyribonucleosidexe2x80x9d refers to deoxyribonucleosides bearing acyl substituents derived from carboxylic acids (which modify the pharmacokinetics and bioavailability of the free deoxyribonucleosides), as disclosed in U.S. Pat. No. 6,297,222, hereby incorporated by reference in its entirety.
The term xe2x80x9csource of at least one deoxyribonucleosidexe2x80x9d or xe2x80x9cdeoxyribonucleoside sourcexe2x80x9d in the context of the subject invention refers to deoxyribonucleosides themselves or derivatives of deoxyribonucleosides which can be converted to deoxyribonucleosides by endogenous enzymes, especially esterases. Examples include acyl derivatives of deoxyribonucleosides (carboxylic acid esters), deoxyribonucleotides (phosphate esters), or oligodeoxyribonucleotides (phosphate diesters). Since esterase activity (involving various enzymes capable of cleaving carboxylic acid esters and phosphate esters) is ubiquitous in mammalian tissues including skin, these esters of deoxyribonucleosides a re converted to deoxyribonucleosides when applied to skin. Similarly, a xe2x80x9csource of at least one ribonucleosidexe2x80x9d refers to a ribonucleoside or ribonucleoside ester, including a ribonucleotide, an oligoribonucleotide, or an acyl derivative of a ribonucleoside.
The term xe2x80x9cester of a deoxyribonucleosidexe2x80x9d (or deoxyribonucleoside ester) refers to either an acyl derivative of deoxyribonucleosides as described above or to a phosphate ester of a deoxyribonucleoside (or deoxyribonucleosides), e.g. deoxyribonucleotides, oligodeoxyribonucleotides, or polydeoxyribonucleotides.
The term xe2x80x9cphotosensitizationxe2x80x9d in the context of the subject invention refers to the process whereby light-absorbing (UV or visible light) molecules directly transfer the energy of an excited state, generally a triplet state, to a target molecule, resulting in damage to DNA and other cellular structures. The target molecule can be DNA itself or another target which results in damage to DNA, e.g. membranes components of lysosomes, which contain deoxyribonuclease.
The term xe2x80x9cphotodynamic sensitizationxe2x80x9d herein refers to the process whereby UV-absorbing molecules generate free radical species or other diffusible reactive intermediates as a result of excitation by UV or visible radiation.
The term xe2x80x9csunscreen agentsxe2x80x9d refers to a UV-absorbing chemicals that are intended to be used in sunscreen products as active ingredients for reducing exposure of the skin to the UV component of solar radiation. Examples of sunscreen agents currently used as such in commercial products include avobenzone (t-butyl dimethoxydibenzoylmethane), oxybenzone (benzophenone-3), dioxybenzone (benzophenone-8), sulisobenzone (benzophenone-4; 2-hydroxy-4-methoxybenzophenone-5-sulfonic acid), octocrylene (2-ethylhexyl-2-cyano-3,3-diphenylacrylate), octyl methoxycinnamate (2-ethylhexyl p-methoxycinnamate), octyl salicylate (2-ethylhexylsalicylate), homosalate (homomenthyl salicylate), trolamine salicylate (triethanolamine salicylate), phenylbenzimidazole sulfonic acid, PABA (para-aminobenzoic acid), roxadimate (ethyl 4-bis hydroxypropyl aminobenzoate), lisadimate (glyceryl PABA), Padimate 0 (octyldimethyl PABA), menthyl anthranilate, and Parsol 1789 (butyl methoxydibenzoyl methane).
The term xe2x80x9cenergy scavengerxe2x80x9d refers to a compound which absorbs energy from the excited states of sunscreen agents or endogenous photosensitizing or photodynamic enhancers of DNA damage, reducing damage to target biological molecules like DNA. In this context, energy scavengers should be nontoxic at effective concentrations and should yield a net reduction in damage to DNA when present during UV irradiation in the presence of a photosensitizer or photodynamic enhancer of DNA damage. In the context of this invention, xe2x80x9cenergy scavengersxe2x80x9d refer particularly to compounds with lowest triplet states with energies less than or equal to those of the nucleobase constituents of genomic DNA. The primary quality of energy scavengers of the invention is that, by virtue of photochemical energy state properties, or mass action (concentration), they prevent damage to genomic DNA that would otherwise occur due to energy transfer for excited chromophores, whether endogenous (e.g. melanin) or exogenous (e.g. sunscreens). Energy scavengers with photochemical properties similar to those of structural constituents of DNA are advantageous, and include sources of at least one deoxyribonculeoside or ribonucleoside, like a deoxyribonucleoside, an acyl deoxyribonucleoside, a deoxyribonucleotide, an oligodeoxyribonucleotide, a ribonucleoside, a ribonucleotide, an oligoribonucleotide, and an acyl ribonucleoside.
The term xe2x80x9cdeleterious consequencesxe2x80x9d as used herein refers to cellular damage in a mammal caused by a mutagen, especially damage to the genome, resulting in an increased chance of developing skin cancer or other skin lesions like solar lentigines, actinic keratoses, or other signs of photoaging like skin wrinkles or xe2x80x9cage spotsxe2x80x9d. Mutagens capable of causing such deleterious consequences include solar radiation, ultraviolet radiation, ionizing radiation, free radicals (whether produced as a result of irradiation of a photochemically active chromophore or from some other source, including normal metabolic processes), nitric oxide, and environmental mutagens.
B. Compounds of the Invention
The compounds of the invention are primarily the major deoxyribonucleoside constituents of DNA: deoxyadenosine, deoxycytidine, deoxyguanosine, and thymidine. The invention also includes the use of effective amounts of precursors of these deoxyribonucleosides, e.g. oligodeoxyribonucleotides, DNA, deoxyribonucleotides and acyl derivatives of deoxyribonucleosides, and, particularly for minimization of effects of photodynamic sensitizers and photosensitizing agents on DNA, ribonucleosides and their congeners, e.g. oligoribonucleotides, ribonucleotides, and acyl derivatives of ribonucleosides.
While not wishing to be bound by a theory, it is believed that the active agents of the invention that pass into cells are the deoxyribonucleosides or acyl derivatives of deoxyribonucleosides, since the anionic phosphate moiety on deoxyribonucleotides or oligodeoxyribonucleotides impedes passage across cell membranes. Phosphorylated deoxyribonucleoside precursors are converted to free deoxyribonucleosides by enzymatic and nonenzymatic degradation before or after application to the skin, prior to their entry into cells.
The deoxyribonucleosides are produced by any of several methods. They are produced by degradation of DNA from biological sources, e.g. fish sperm, by chemical synthesis, or by fermentation technology.
Also encompassed by the invention are pharmaceutically acceptable salts of the above-noted compounds.
C. Compositions of the Invention
The invention includes pharmaceutical compositions for improving the net fidelity of DNA repair and for protecting the skin against mutagens. The composition comprises 1) an effective amount of a source of one or more deoxyribonucleosides, and optionally 2) an effective amount of a pharmaceutically acceptable topical carrier capable of delivering the deoxyribonucleosides or their precursors to appropriate target cells in the skin under in vivo conditions.
While individual deoxyribonucleosides, especially deoxycytidine (see Example 7) have some activity in attenuating UV-induced tumorigenesis, two or more deoxyribonucleosides, or preferably all four, are typically included in a formulation of the invention. Encompassed by the invention are compositions containing deoxyadenosine, deoxycytidine, deoxyguanosine, or thymidine, either as single agents, or in all possible combinations of two, three, or all four of these compounds. Compositions containing deoxycytidine are particularly advantageous. The concentrations of individual deoxyribonucleosides in compositions encompassed by the invention, whether present individually or in combination with other deoxyribonucleosides or deoxyribonucleoside precursors (such as deoxyribonucleoside esters), and normalized to the amount of free nucleoside or nucleoside moiety in the case of deoxyribonucleoside phosphates or oligonucleotides or prodrugs like acylated deoxyribonucleoside derivatives, range from 0.1 to 10 mg/ml, advantageously 1 to 5 mg/ml.
In order to permit access of the deoxyribonucleosides and related compounds of the invention to deeper-lying skin cells, vehicles which improve their penetration through the outer layers of the skin, e.g. the stratum corneum, are useful. Vehicle constituents which improve the penetration of compounds of the invention into the skin include but are not limited to: ethanol, isopropanol, azone (1-dodecylazacycloheptan-2-one), oleic acid, linoleic acid, propylene glycol, hypertonic concentrations of glycerol, lactic acid, glycolic acid, citric acid, and malic acid.
In addition to promoting absorption of agents into the skin, use of topical alpha-hydroxy acids (AHA), e.g. lactic acid and glycolic acid, can affect the ability of the skin to reduce the penetration of ultraviolet light into the vulnerable basal layers of the epidermis. Thus, there is also an increased need for agents which reduce the consequences of exposure to solar or ultraviolet radiation in people using AHA""s, e.g. for promoting exfoliation of epidermal cells. Since penetration of UV-absorbing sunscreen agents into the skin is undesirable because of possible photosensitization and photodynamic enhancement of UV-induced damage to cells, the compounds of the invention are uniquely suitable for combination with AHA""s, either in the same formulation or a separate one, for improving skin resistance to damaging effects of solar or ultraviolet radiation.
One embodiment of the invention is a hydrogel formulation, comprising an aqueous or aqueous-alcoholic medium and a gelling agent, and a deoxyribonucleoside source. Suitable gelling agents include but are not limited to methylcellulose, carboxymethylcellulose, hydroxypropylmethylcellulose, carbomer (carbopol), hypan, polyacrylate, and glycerol polyacrylate.
Liposomes are microscopic lipid vesicles which can contain pharmacologically active agents either enclosed in the aqueous space within the vesicle or in the lipid membrane itself, depending on the lipophilicity of the agent. Liposomes are capable of delivering a pharmacologic agent through the stratum corneum into deeper layers of the skin, and are therefore suitable vehicles for compounds and compositions of the invention.
Niosomes are lipid vesicles similar to liposomes with membranes consisting largely of non-ionic lipids, some forms of which are effective for transporting compounds across the stratum corneum.
In one embodiment of the invention, lipophilic acyl derivatives of deoxyribonucleosides, e.g. oleic or palmitic acid esters of deoxyribonucleosides are incorporated into membranes of niosome or liposome membranes, in addition to or instead of being enclosed within the vesicular membranes.
Other agents which are advantageous for incorporation into a composition of the invention include corticosteroids, especially hydrocortisone in concentrations of 0.05 to 1%, other anti-inflammatory corticosteroids at therapeutically effective concentrations, topical anesthetics including but not limited to benzocaine, lidocaine, and benzyl alcohol, aloe vera and aloe barbadensis, retinoids, antioxidants like Vitamins C and E, flavins, polyphenols (e.g. extracted from green tea or black tea), allantoin, liposomal DNA repair enzymes, antibacterial agents (e.g. quaternary ammonium compounds, bacitracin, neomycin, polymyxin), zinc salts, and methylxanthines. All of these listed agents have some utility in treating or attenuating various aspects of skin injury or discomfort caused by ultraviolet radiation or inflammatory skin conditions, and are therefore complementary to the unique actions of the deoxyribonucleosides of the invention.
Benzyl alcohol, which is known to have anesthetic and preservative properties, has the unexpected effect of improving aqueous solubility of the relatively insoluble purine deoxyribonucleosides, deoxyadenosine and deoxyguanosine; preferred concentrations of benzyl alcohol in topical formulations of deoxyribonucleosides are 0.5 to 5%. This is very important in permitting high concentrations of the deoxyribonucleosides of the invention to be stably incorporated into aqueous vehicles.
Sunscreens
The compounds of the invention are advantageously incorporated into the same formulation as a UV-absorbing chemical sunscreen agent such as: avobenzone (t-butyl dimethoxydibenzoylmethane), oxybenzone (benzophenone-3), dioxybenzone (benzophenone-8), sulisobenzone (benzophenone-4; 2-hydroxy-4-methoxybenzophenone-5-sulfonic acid), octocrylene (2-ethylhexyl-2-cyano-3,3-diphenylacrylate), octyl methoxycinnamate (2-ethylhexyl p-methoxycinnamate), octyl salicylate(2-ethylhexylsalicylate), homosalate (homomenthyl salicylate), trolamine salicylate (triethanolamine salicylate), phenylbenzimidazole sulfonic acid, PABA (para-aminobenzoic acid), roxadimate (ethyl 4-bis hydroxypropyl aminobenzoate), lisadimate (glyceryl PABA), Padimate O (octyldimethyl PABA), menthyl anthranilate, or Parsol 1789 (butyl methoxydibenzoylmethane).
Alternatively, the compounds of the invention are formulated in a base which is suitable for application to the skin prior to, or after, application of a sunscreen. This embodiment of the invention permits the benefits of deoxyribonucleosides, in reducing consequences of UV damage and in attenuating photodynamic enhancement of cellular damage caused by sunscreen agents themselves, to be obtained in conjunction with use of a wide variety of commercial sunscreen products.
D. Therapeutic Uses of the Compounds and Compositions of the Invention
Reduction of Deleterious Consequences of Exposure of Skin to Ultraviolet or Solar Radiation
The compounds and composition of the invention, when applied or administered before, during, or after exposure of the skin of a mammal to mutagenic radiation, have the unexpected activity of improving the net fidelity DNA repair, thereby reducing mutation frequency, photoaging, and the chance of developing skin cancers.
DNA repair proceeds by several steps. A chemical lesion in DNA, which can be caused by ultraviolet radiation, ionizing radiation, free radicals, or chemical mutagens, is detected by the DNA repair system. A segment of nucleotides including the damaged region is excised, generally along with a number of surrounding nucleotides. The excised strand is then resynthesized from free deoxyribonucleotides, using the intact DNA strand as a template.
It is generally believed that improvement of repair of DNA in skin cells (e.g. keratinocytes, melanocytes, fibroblasts) would require alteration in the activity of enzymes or other proteins involved in the detection and excision of DNA lesions. Thus, there have been attempts to improve DNA repair in skin by delivering DNA repair enzymes via topically-applied liposomes (U.S. patents (Yarosh et al., Cancer Res. 52:4227-31, 1992).
Far more important than the initial rate of excision of lesions is the fidelity or accuracy of repair. Agents which accelerate the excision step of DNA repair can actually exacerbate damage if the cells are incapable of accurate repair synthesis at a rate that matches the rate of excision of damaged segments of DNA (Collins and Johnson, J. Cell Physiol. 99:125-137, 1979).
Compounds and compositions of the invention, when applied to skin before, during or after exposure to solar or ultraviolet radiation or other environmental mutagens, reduces the mutation frequency otherwise induced by the mutagen (Example 1). This reduces the damage to cells caused by the mutagen, and reduces the chance of development of skin cancers, as shown in Examples 2 and 7. The effect on genomic fidelity is in principle mediated by actual improvement in the fidelity of repair in an individual cell or by improvement of the elimination of cells with irreparable DNA damage. Either mechanism results in a net improvement in genomic fidelity in skin exposed to mutagens.
The compounds of the invention have unanticipated benefits when used in combination with other agents known to be useful in various aspects of skin care, including but not limited to sunscreens, methylxanthines, retinoids, DNA repair enzymes, exfoliants, and protease inhibitors, corticosteroids and nonsteroidal anti-inflammatory agents.
The compounds and compositions of the invention, when applied soon enough, e.g. within about 3 days after exposure to ultraviolet or solar radiation, improve the repair of cellular and macromolecular damage and improve net genomic fidelity, thereby reducing the chance of development and severity of macroscopically visible deleterious consequences of such exposure, including but not limited to photoaging, sunburn symptoms, actinic keratoses, solar lentigines, xe2x80x9cage spotsxe2x80x9d, and skin cancer. The compounds and compositions of the invention are also optionally applied before or during exposure to solar or ultraviolet radiation to shorten the time gap between damage and onset of repair enhancement by the compounds of the invention.
Treatment of skin with compounds and compositions of the invention results in a reduced chance of development of skin cancers and other deleterious consequences of exposure to solar or UV radiation like photaging even when the compounds of the invention are applied even after irradiation, e.g. after unintended exposure to potentially-damaging doses of solar radiation. This type of activity is not shared by conventional sunscreens or agents which might act by enhancing melanogenesis, which are useful only if applied before irradiation.
Improvement of Sunscreen Activity and Attenuation of Photodynamic Enhancement of UV Damage by Sunscreens
Sunscreens are typically designed and tested on the basis of prevention of sunlight-induced erythema. While erythema and its attenuation by sunscreens is an important short-term effect, reduction of erythema and inflammation by sunscreens does not necessarily mean that they produce a proportionate protection of DNA (or prevention of skin cancers and some features of photoaging secondary to DNA damage). Sunscreens are certainly useful in preventing some manifestations of photoaging and UV-related carcinogenesis, but do not provide complete protection, and in some situations may actually exacerbate photoinjury by acting as photodynamic sensitizers (see Example 3).
Chemical sunscreens are intended to act by absorbing photons at mutagenic (or erythmogenic) wavelengths, thus producing a short-lived excited singlet state; return to the ground state is accompanied by photon emission at longer wavelengths that are supposed to be less harmful than the incident radiation. Photon emission during the rapid return of a molecule from an excited singlet state to a ground state is known as xe2x80x9cfluorescencexe2x80x9d. However, sunscreens or other exogenous or endogenous UV-absorbing molecules can also be excited to longer-lived triplet states which can facilitate further reactions (the energy-emission that occurs during return of a molecule from an excited triplet state to a ground state is known as xe2x80x9cphosphorescencexe2x80x9d, and typically occurs over a much longer time span than fluorescence). The consequence is that some UV-absorbing agents, especially those with a lowest triplet state that has a higher energy level than the lowest triplet state of genomic DNA constituents, can absorb photons and actually exacerbate damage to DNA by direct or indirect energy transfer (e.g. from a triplet excited state) rather than by simple fluorescence, or photon emission at harmless wavelengths.
Benzophenone, a close structural analog of oxybenzone, increases the yield of strand breaks and pyrimidine dimers in UV-irradiated DNA, and is known to produce free radicals upon irradiation with UV light (Charlier et al., Photochemistry and Photobiology, 15:527-536, 1972). The results presented in Examples 3, 4, 5 and 6 indicate that a similar phenomenon also occurs with approved sunscreen ingredients, and that the deoxyribonucleosides of the invention attenuate this deleterious consequence of sunscreen use.
When present in concentrations sufficient to block access of UV radiation to cells, sunscreen agents are protective. However, if present in very low concentrations, insufficient to adequately block UV transmission, photon-absorbing agents, including common sunscreen ingredients, can operate as energy-transfer molecules, efficiently trapping UV energy and transferring it to cell components, either directly (photosensitization) or by catalyzing formation of reactive oxygen radicals (photodynamic sensitization). Thus, low concentrations of oxybenzone, for example, enhance DNA damage induced by ultraviolet radiation, whereas higher concentrations, sufficient to block UV access to the cells or their immediate microenvironment altogether, protect against DNA damage (see Example 3).
The expected activity in vivo is that an oxybenzone-containing sunscreen would protect cells from damage if present in a layer sufficient to block access of light to the target cells altogether. However, at lower concentrations, insufficient to prevent penetration of UV radiation to target cells, and especially if some oxybenzone has been absorbed into the critical cell layers, either via passage through the stratum corneum or through hair follicles, there may be potentiation of damage to DNA in vivo. In mice treated topically with commercial sunscreen containing oxybenzone, effects consistent with this hypothesis are in fact observed after exposure to UV (see Example 6). Classes of sunscreens other than benzophenone derivatives also exacerbate UV-induced damage to DNA when present at low concentrations during irradiation.
A strain of transgenic mice (v-HA-ras transgenic TG.AC mice) which is very susceptible to a variety of carcinogens, including UV radiation has been developed recently (Leder et al., Proc. Nat. Acad. Sci. USA, 87:9178-9182, 1990). In response to a relatively small exposure to UV radiation, these mice reliably develop cutaneous papillomas within a few weeks. When a circular patch of commercial sunscreen is applied to the back of such a mouse, the center of the protected region does in fact have lower incidence of UV-induced papillomas than the unprotected side, but often, along the margin of the applied sunscreen, there is a very high incidence (sometimes higher than in unprotected areas) of papillomas (See Example 6). A layer of sunscreen sufficient to block UV access to target cells is protective, but low concentrations, e.g. at the margin of a patch of sunscreen) can act as photosensitizers increasing the incidence of a UV-induced skin cancer beyond that seen in completely xe2x80x9cunprotectedxe2x80x9d skin. Exposure of relevant skin cells to low photosensitizing (rather than protective) concentrations of sunscreens clearly must occur during ordinary usage, e.g. at the margin of an applied patch, or as a protective layer is washed or worn off.
A benefit of deoxyribonucleosides or related compounds added to conventional sunscreen formulations (or other cosmetics containing sunscreens), beyond the support of DNA repair, is to synergize with conventional sunscreen compounds by acting as energy scavengers which trap energy emitted by (or radicals produced by) sunscreen agents that is chemically similar to the cellular target, DNA. Exogenous deoxyribonucleosides (in addition to their direct absorbance of UV energy) serve as xe2x80x9cdecoysxe2x80x9d for energy captured by sunscreen agents or other photosensitizers that would otherwise be transferred to cellular targets, including DNA. Thus, deoxyribonucleosides provide a dose-dependent reduction in damage caused to cellular DNA by UV radiation in the presence of low concentrations of photosensitizing agents like oxybenzone or other sunscreen agents (Example 4).
A defining characteristic of suitable energy scavenging agents is that their lowest triplet energy state is equal to or lower than that of DNA constituents in situ. Because of the similarity of physicochemical properties of deoxyribonucleosides (or deoxyribonucleotides, acyl deoxyribonucleosides, or oligodeoxyribonucleotides) and genomic DNA, the compounds of the invention are particularly suitable as energy-scavenging agents to protect genomic DNA from damage due to energy transfer from photosensitizers or photodynamic sensitizers. In this embodiment, ribonucleosides, ribonucleotides, oligoribonucleotides and acyl derivatives of ribonucleosides are within the scope of the invention. Appropriate concentrations of such scavengers in a composition for topical application range from 0.1 to 100 milligrams per milliliter (normalized to the amount of free nucleoside present in the case of nucleotides or oligonucleotides or acyl derivatives of nucleosides). Advantageously, such scavengers are present in concentrations ranging from 0.1 to 20 mg/ml or especially 1 to 5 mg/ml.
The problem of photodynamic enhancement of damage to DNA extends beyond sunscreens. Other compounds including endogenous molecules in the skin, can absorb UV radiation at wavelengths that do not necessarily directly damage DNA significantly, and transfer that energy to cellular targets including DNA, or generate free radicals that damage cellular DNA. Examples of endogenous photosensitizing or photodynamically active skin constituents (photochemically active chromophores) include but are not limited to porphyrins, tryptophan, riboflavin, and melanin. Exogenous photodynamically active compounds include psoralens, which are present in some perfume oils (bergamot), and which are in fact used to enhance sunlight-induced tanning and UV phototherapy of psoriasis through exacerbation of cellular injury. Many therapeutic drugs or their metabolites are photochemically active chromophores which can produce skin adverse reactions when a patient is exposed to solar, visible, or ultraviolet radiation. Pigments and other light-absorbing constituents of cosmetics are also photochemically active chromophores which can exacerbate cellular photodamage.
The deoxyribonucleosides and related compounds of the invention (e.g. deoxyribonucleotides, oligonucleotides, or DNA itself) are useful for attenuating cellular damage caused by excited light-absorbing molecules, including exogenous photochemically active chromophores like sunscreens and cosmetic pigments, and also from endogenous chromophores like tryptophan, porphyrins, urocanic acid and melanin.
Furthermore, since the photodynamic enhancement of DNA damage caused by benzophenone derivatives is in part mediated by production of free radicals (Charlier et al., Photochemistry and Photobiology, 15:527-536, 1972), compounds and compositions of the invention are useful for protecting the skin and mucosa from free radical damage, whether or not the free radicals (e.g. hydroxyl radicals, peroxide radicals, or lipoperoxide radicals) are initiated or produced by photodynamic phenomena. Examples 3,4,5 and 6 provide evidence that the deoxyribonucleosides of the invention protect against DNA caused by free radicals.
The deoxyribonucleosides and related compounds of the invention are advantageously incorporated into the same formulation as chemical sunscreen agents, which include but are not limited to: avobenzone (t-butyl dimethoxydibenzoylmethane), oxybenzone (benzophenone-3), dioxybenzone (benzophenone-8), sulisobenzone (benzophenone-4), octocrylene (2-ethylhexyl-2-cyano-3,3-diphenylacrylate), octyl methoxycinnamate (2-ethylhexyl p-methoxycinnamate), octyl salicylate (2-ethylhexylsalicylate), homosalate (homomenthyl salicylate), trolamine salicylate (triethanolamine salicylate), phenylbenzimidazole sulfonic acid, PABA (para-aminobenzoic acid), roxadimate (ethyl 4-bis hydroxypropyl aminobenzoate), lisadimate (glyceryl PABA), Padimate O (octyldimethyl PABA), menthyl anthranilate, or Parsol 1789 (butyl methoxydibenzoylmethane). Such sunscreen agents are present in formulations at concentrations that are in accord with regulatory guidelines and standard use.
Similarly, in another embodiment of the invention, compounds of the invention are incorporated into cosmetics containing photochemically active chromophores to minimize deleterious consequences of combined exposure of skin to solar or ultraviolet radiation and such cosmetic ingredients.
Alternatively, the compounds of the invention are applied to the skin in a separate composition, e.g. a spray, lotion, roll-on, stick, or gel, before or after a sunscreen product or cosmetic is applied.
Methylxanthines
Methylxanthines, such as caffeine, theophylline, aminophylline or isobutylmethylxanthine, have been proposed as xe2x80x9csunlessxe2x80x9d tanning agents, which act by modulating activity of the biochemical pathways involved in melanogenesis. Their proposed mechanism involves inhibition of cyclic-AMP phosphodiesterase, thus enhancing the biological activity of cyclic AMP in the pathways regulating melanogenesis. Methylxanthines can enhance the production of melanin in melanocytes, acting either alone or in combination with tanning stimulants like ultraviolet or solar radiation. However, methylxanthines are also known to exacerbate DNA damage caused by ultraviolet radiation, which has been attributed to an impairment of DNA repair or disruption of cell cycle control mechanisms (Kastan et al., Cancer Research, 51:6304-6311, 1991).
Compounds of the invention are useful for reducing the deleterious effect of methylxanthines on DNA damage caused by exposure of skin to ultraviolet or solar radiation. The compounds of the invention thus improve the safety of skin tanning products that contain methylxanthines as active ingredients. Compounds of the invention are applied either separately or in the same formulation as the methylxanthines.
Exfoliants
Cosmetics containing alpha-hydroxy acids (AHA) such as lactic acid, glycolic acid, citric acid, or malic acid are widely used. They have moisturizing and exfoliant properties. Products containing high concentrations of AHA are also used to produce more extreme xe2x80x9cskin peelsxe2x80x9d, in which the outer layers of the epidermis are removed, essentially by means of a chemical burn. New epidermis growing in is often softer and smoother than the skin layers that were removed. Beta-hydroxy acids like salicylic acid are also useful exfoliants. Retinoic acid is used for similar purposes through its exfoliant actions and through stimulation of epidermal cell turnover and alteration of epidermal metabolism.
Exfoliants are reported to reduce the sun-blocking capabilities of the stratum corneum, and furthermore increase the permeability of the skin to other agents. Thus, there is a need for use of UV protection in conjunction with AHA""s, yet the increased skin permeability produced by AHA requires caution in the selection of sun protection agents, since absorbed sunscreen agents can produce photodynamic enhancement of DNA damage. The compounds of the invention are effective in reducing deleterious consequences of exposure to sunlight or ultraviolet radiation or other environmental mutagens in subjects using exfoliants, including but not limited to alpha-hydroxy acids, beta-hydroxy acids, and retinoids.
Nonsteroidal Anti-inflammatory Agents
Nonsteroidal anti-inflammatory agents are commonly used for treatment of arthritis and other anti-inflammatory agents. Moreover, some members of this class, e.g. diclofenac (2,6-dichloro-phenyl-amino-phenylacetate) are under investigation as topical agents for reducing some aspects of skin photodamage.
One of the prototypical members of this class of drugs, acetaminophen, inhibits DNA repair after damage caused by UV radiation by inhibiting the enzyme ribonucleotide reductase, which converts ribonucleoside diphosphates to deoxyribonucleoside diphosphates (Hongslo et al., Mutagenesis, 8:423-429, 1993). By supplying deoxyribonucleosides to skin, especially to areas of the skin that are generally exposed to sunlight, of patients receiving either oral or topical treatment with nonsteroidal anti-inflammatory agents, compositions of the invention overcome a deleterious consequence associated with this widely-used class of drugs.
Ornithine Decarboxylase Inhibitors
One consequence of UV irradiation of skin is an increase in cell proliferation rate and in the activity of enzymes necessary for cell proliferation, one of which is ornithine decarboxylase (ODC). Difluoromethylornithine (DFMO) is an inhibitor of ODC, which is necessary for polyamine synthesis, which in turn is necessary for DNA replication. DFMO is useful for reducing the incidence of skin cancer and precancerous actinic lesions. Inhibition of cell cycling after exposure to solar or UV radiation may give cells more time to repair DNA before mutagenic lesions are fixed by cell division, but cell cycle stasis also generally results in reduced levels of deoxyribonucleosides, which are necessary for repair of DNA damage. Deoxyribonucleosides are therefore useful in conjunction with DFMO and other antiproliferative agents which act via mechanisms not directly involving induced depletion or imbalance of deoxyribonucleotide pools. Deoxyribonucleosides of the invention are optionally incorporated into the same formulation as DFMO (or other inhibitors of skin cell proliferation that act through mechanisms other than impairment of DNA precursor synthesis), or are applied in a separate formulation.
Treatment of Skin During Exposure to Endogenous Nitric Oxide
Nitric oxide (NO) is a biologically active mediator released by endothelial cells, macrophages and other cell types, especially during inflammatory episodes. Nitric oxide is also an important mediator of erythema associated with UV exposure. Inhibitors of NO synthetase attenuate the increase in skin blood flow following exposure (Deliconstantinos et al., J Cardiovasc Pharmacol 20 Suppl 12:S63-5, 1992; Deliconstantinos et al., Br J Pharmacol 114(6):1257-65, 1995; Warren, FASEB Journal, 8(2):247-51, 1995).
Nitric oxide is a potent inhibitor of the enzyme ribonucleotide reductase (RR), which is the key enzyme for de novo synthesis of deoxyribonucleotides (Kwon et al., J Exp Med 174(4):761-7, 1991; LePoivre et al., J Biol Chem 269(34):21891-7, 1994). The antiproliferative effects of nitric oxide are in part attributable to inhibition of ribonucleotide reductase. This can be beneficial in an inflammatory response to an infectious microorganism or a neoplasm, but is deleterious in cells in need of capability for DNA repair, e.g. skin exposed to inflammatory mediators elicited by UV exposure. The amounts of NO released from macrophages are sufficient to inhibit ribonucleotide reductase and thereby induce cytostasis in neighboring cells.
The best-known inhibitor of RR, hydroxyurea (HU), has structural similarities to N-omega-hydroxy-1-arginine, a physiological intermediate in NO production. Hydroxyurea can act as an NO-like nitrosating reactant (LePoivre et al., J Biol Chem 269(34):21891-7, 1994). Both NO and hydroxyurea inhibit RR by quenching a tyrosyl radical in the active site of the enzyme.
A discovery first disclosed herein is that NO sensitizes cells to UV-induced DNA damage via mechanisms that are reversible with exogenous deoxyribonucleosides (see Example 8). Moreover, NO by itself, in the absence of UV exposure, causes DNA damage that is prevented or reversed by compounds of the invention (Example 8).
The inflammatory response to UV exposure (which also plays an important role in UV-associated immunosuppression), via inhibition of ribonucleotide reductase by NO, exacerbates the mutagenic and cytotoxic effects of UV exposure. The subject invention provides a means for compensating for this deleterious effect of NO. Exogenous deoxyribonucleosides, which enter into DNA metabolism downstream of ribonucleotide reductase, compensate for reduced RR activity. NO, although deleterious in cells needing deoxyribonucleotides, has some beneficial effects in sun-exposed skin. Inhibitors of NO production also impair the healing of skin damaged by exposure to excessive UV radiation, i.e., UV doses that produce a sufficiently severe sunburn for wound healing processes to be called into action (Benrath et al., Neurosci Lett, 1995). Thus, NO is useful for tissue repair (probably by improving blood flow and perhaps also by stimulating growth factor release from macrophages or keratinocytes), but may simultaneously exacerbate UV-related damage to DNA by reducing cellular capacities for production of deoxyribonucleotides for DNA repair.
NO-mediated inhibition of RR provides a mechanism for a disproportionate increase in DNA damage without a corresponding increase in other symptoms of sun exposure during repeated exposure to strong sunlight. Topical application of the compounds and compositions of the invention provides a method for ameliorating this form of conditional hypersensitivity.
NO participates in skin inflammatory reactions that do not necessarily involve exposure to solar or ultraviolet radiation. NO, which is released from activated macrophages, is component of most inflammatory reactions. The compounds and methods of the invention provide a means of overcoming some deleterious effects of NO in inflammatory skin conditions, including but not limited to psoriasis, dermatitis, allergic dermatitis, eczema and acne. In these conditions, NO sensitizes some cell types to UV-induced DNA damage by inhibiting deoxyribonucleotide synthesis. Compounds and compositions of the invention ameliorate this deleterious consequence of combined UV exposure and inflammatory skin conditions. Since NO and other endogenous oxidants that cause DNA damage are present in inflammatory skin conditions like psoriasis or dermatitis even in the absence of significant exposure to UV radiation, the deoxyribonucleosides of the invention are useful for protecting genetic integrity of skin cells in inflammatory conditions. Compounds of the invention prevent or repair DNA damage caused by NO alone, without exposure to UV radiation (Example 8).
Ribonucleosides are also useful for treating inflammatory skin an mucosal conditions, in part by providing higher concentrations of the ribonucleotide substrates for ribonucleoside reductase. Ribonucleosides (or ribonucleoside esters) in this context are used in the same ways that deoxyribonucleosides, advantageously in topically-applied compositions, with ribonucleosides (or ribonucleoside esters) incorporated in concentrations ranging from 0.1 to 20 mg/ml, advantageously 1 to 5 mg/ml. Adenosine is particularly useful for treatment of inflammatory conditions.
Inhibition of DNA precursor synthesis by hydroxyurea leads to enhanced activity and leakage of hydrolytic lysosomal enzymes which participate in extracellular damage, e.g. in inflammatory skin conditions or photodamage (Malec et al., Chem. Biol. Interact., 57:315-324, 1986). The compounds of the invention prevent this component of inflammatory tissue injury, especially when such inhibition of DNA precursor synthesis is mediated by endogenously-produced NO, which is functionally similar to hydroxyurea.
A further contribution to genomic damage is that exposure of cells to ultraviolet radiation results in the release of enzymes, including deoxyribonuclease, from lysosomes. Deoxyribonuclease II, which is present in lysosomes, is an endonuclease that can produce strand breaks in nuclear DNA following lysosome disruption. Leakage of enzymes from lysosomes also occurs in inflammatory conditions in general. The compounds and compositions of the invention are useful for preserving genomic integrity after chromosomal damage caused by deoxyribonuclease released from lysosomes in inflammatory skin conditions and after exposure of skin to solar or ultraviolet radiation.
By limiting some of the deleterious consequences of skin inflammation, compounds and compositions of the invention are useful as anti-inflammatory agents, and are optionally administered (either as separate compositions or, advantageously, in the same formulation) in conjunction with other topical or systemic anti-inflammatory agents including but not limited to corticosteroids like hydrocortisone and its congeners.
Similarly, compounds and compositions encompassed by the invention are useful for treatment of mucosal inflammatory conditions, including but not limited to inflammatory bowel disease, ulcerative colitis, or Crohn""s disease, or mucositis anywhere in the gastrointestinal tract. The preferred mode of treatment is by topical administration, in this case via enema or suppository, for which purposes deoxyribonucleosides or other compounds of the invention are incorporated into suitable vehicles.
Treatment of Skin and Mucosal Tissues Exposed to Ionizing Radiation
Patients receiving therapeutic treatment (e.g. for cancer) with ionizing (X-Ray or gamma) radiation can suffer damage to skin overlying an internal tumor, leading to desquamation and poor healing. Compounds and compositions of the invention are useful for treating damage to skin and mucosal surfaces caused by intentional or accidental exposure to ionizing radiation. For treatment of skin, compositions of the invention are applied topically before or after radiation treatment. For treatment of mucosal surfaces, e.g. in the mouth, gastrointestinal tract, urethra or vagina, appropriate compositions of the invention are also applied topically. Suitable compositions for treatment of mucosal surfaces include gels, lotions, ointments, suppositories, orally-administered capsules, pills or dragees, or solutions.
E. Administration and Formulation of Compounds and Compositions of the Invention
Compounds of the invention are formulated in pharmaceutically acceptable vehicles that deliver the compounds to the necessary cell populations in skin at concentrations adequate to accomplish the objectives of reducing mutation frequency and chance of developing cancer.
Compositions of the invention are applied before, during, or after exposure to sunlight or other mutagens. A lotion or hydrogel containing deoxyribonucleosides (0.1 to 10 mg/ml, advantageously 1 to 5 mg/ml) is applied to skin as a thin film. The composition should be applied within about 48 or 72 hours after exposure to damaging doses of sunlight or ultraviolet radiation, in order to provide support for DNA repair prior to the first cell divisions after irradiation, although application before, during, or within 12 hours after exposure to intense sunlight is advantageous. Compositions of the invention are also effective when applied before exposure to radiation or other mutagens, as long as the deoxyribonucleosides so provided, or their anabolites, are available to cells in need of their beneficial effects at the time of exposure to a mutagen. Advantageously, compositions of the invention are applied within about 12 hours before exposure of the skin to a solar radiation or other mutagens.
Compositions of the invention are advantageously applied as a daily-use skin treatment, once to several times per day, especially on sun-exposed parts of the body, or sites of inflammatory skin conditions. Exposure to solar radiation leading to skin photodamage and photoaging is generally a cumulative process, involving repeated exposure to sunlight, even daily, over a period of years. In this context, use of the compounds and compositions of the invention to prevent or treat photodamage to the skin involves treatment of existing lesions due to prior sun exposure, as well as prevention of, or attenuation of the severity of, damage due to present and future exposure to sunlight or other mutagens.
By improving repair of molecular damage to DNA as it occurs or before it is permanently established in the genome by cell division, or by preventing initial damage through energy scavenging, compositions of the invention prevent or delay the manifestation of deleterious consequences of radiation, free radicals, or chemical mutagens, such as grossly visible skin damage, photoaging, actinic keratoses, and skin cancer. Thus, compositions and methods of the invention reduce the rate of appearance and the incidence of signs of skin photodamage, especially when administered regularly, e.g. daily, or especially before, during, or after exposure to solar radiation.
In one embodiment of the invention, a composition containing deoxyribonucleosides is applied to skin prior to application of a sunscreen, as an alternative to use of formulations containing both conventional sunscreens and deoxyribonucleosides or related compounds of the invention.
For treatment of colon mucosal inflammation, e.g. inflammatory bowel disease, compositions of the invention are administered as a suppository or enema, approximately once per day according to clinical need. Volumes of 10 to 500 ml of a suitable enema composition containing deoxyribonucleosides are suitable for treatment of inflammatory bowel disease, e.g. ulcerative colitis or Crohn""s disease. For treatment of mucositis in other parts of the gastrointestinal tract, such as the mouth, standard pharmaceutically acceptable vehicles for that route of administration are used, e.g. mouthwashes or adherent hydrocolloids.
F. Synthesis of the Compounds of the Invention
Deoxyribonucleosides, being constituents of DNA, are present in all living organisms, and can therefore in principle be extracted from a variety of sources. In practice, the most convenient biological sources at present are fish milt, which contains a relatively high concentration of DNA. Fish milt sacs are homogenized, and the DNA therein is partially purified and treated with deoxyribonucleases and phosphatases to degrade it to the level of nucleosides, which are then purified by chromatography and recrystallization.
Since mixtures of deoxyribonucleosides are used in some embodiments of the invention, purified deoxyribonucleosides are recombined in appropriate proportions. Alternatively, deoxyribonuclosides are not separated from each other during purification from other fish milt components (or other contaminants if the DNA is derived from other biological sources); appropropriate quantities of individual deoxyribonucleosides are added to such a mixture, if necessary, to adjust the relative proportions of deoxyribonucleosides.
Deoxyribonucleosides can also be synthesized chemically from simpler precursors.
Acyl derivatives of deoxyribonucleosides, as disclosed in U.S. Pat. No. 6,297,222, are useful for 1) providing sustained availability of deoxyribonucleosides due to gradual deacylation by nonspecific esterases in the skin, and 2) improved penetration through hydrophobic biological membranes or extracellular media, e.g. the intercellular lipids in the stratum corneum of the epidermis.
It will be obvious to the person skilled in the art that other methods of synthesis may be used to prepare the compounds of the invention.

Antimutagenic compositions for treatment and prevention of photodamage to skin


The invention relates to a process for removal of sulfur compounds from wastewater, which process provides recovery of elemental sulfur. The process is carried out in two separate anaerobic reactors. In the first reactor organic compounds are converted to acid compounds and sulfur compounds are converted to sulfide compounds. The sulfide compounds are stripped from said first reactor with a stripping gas. Subsequently, the effluent of the first reactor is fed to a second anaerobic reactor, in which organic compounds, such as the acid compounds, are converted to produce biogas, while any unconverted sulfur compounds from the previous step are converted to sulfide compounds as well. Finally, the sulfide compounds are removed from the second reactor in a stripping system, and converted to elemental sulfur in an adsorber.

The invention relates to a process for anaerobic removal of sulfur compounds from wastewater.
Anaerobic biological processes for the treatment of wastewater having a high COD (chemical oxygen demand) value are known in the art. In such known processes, organic compounds are converted to biogas (a gas mixture comprising CO2 and CH4) at temperatures above about 25xc2x0 C. When wastewater contains high amounts of sulfur compounds (e.g., more than ca. 100 mg S/dm3), such as sulfates, problems may occur in operating such anaerobic reactors due to the presence of sulfide, which is formed under anaerobic conditions from sulfate. Sulfide may inhibit the methane formation. Also sulfide will give rise to H2S formation at pH less than 9. H2S is a toxic and corrosive gas and requires measures to control odour. The sulfide may be reoxidized into sulfate, but this requires an additional aerobic step. Furthermore, regulations often impose a strong limitation on the amount of sulfur compounds that can be discarded into the environment.
As a consequence, there is a need for processes to remove sulfide from wastewater. In the art different approaches have been suggested for sulfide removal processes. For example, EP-A-0 766 650 describes an anaerobic process in which H2S is stripped from a methanogenic reactor using a stripping gas. The H2S rich stripping gas is fed to a scrubber where the H2S is converted into elemental sulfur. The scrubber is operated with a regenerable redox liquid that contains an iron(III) chelate or an iron(III) complex. During this absorption process sulfide is converted to elemental sulfur, while Fe(III) is reduced to Fe(II). Fe(II) is reoxidized to Fe(III) by air in a separate aerator. This known process is particularly suited for the treatment of alkaline wastewater, such as tannery wastewater.
However, the known processes have a number of drawbacks.
The anaerobic reactor in known processes is operated at a relatively high pH (8-8.5), in particular when alkaline wastewater is treated having a pH of 9-12. As a result, the stripping of H2S is slow, since H2S dissolves more easily in water at higher pH. To compensate for this, equipment of a large volume has to be employed, in particular the stripper column, which has to be operated with large volumetric flows. An example of such a process employing an external stripper is found in U.S. Pat. No. 5,500,123. Alternatively, acid, such as formic acid, can be added. Both alternatives bring about high equipment and/or operating costs. Another result of operating the anaerobic reactor at a relatively high pH is that carbonate may precipitate in the stripper, which leads to fouling and clogging of the equipment.
When the H2S loaded gas is scrubbed in the known processes, inevitably an amount of CO2, which is present in the stripped gas as well, is absorbed in the scrubber liquid. This CO2 is eventually vented when the scrubber liquid is regenerated. This net removal of CO2 from the system gives rise to a further increase of the pH. Moreover, the formation of carbonate salts due to the presence of CO2 in the stripper equipment may give rise to fouling and clogging. The precipitation of carbonate salts may be prevented by lowering the pH, however, this lowers the rate of absorption and reaction of H2S, as a result of which larger absorbers and volumetric flowrates are required.
These drawbacks become apparent for example in the process of EP-A-0 766 650, where the aqueous effluent needs to be recirculated over the stripper a number of times in order to lower the sulfide concentration, since per pass only a small amount of sulfide is stripped. This is the result of the unfavourable equilibrium at higher pH values. This recirculation results in further disadvantages, since the anaerobic reactor is operated at a higher hydraulic loading rate, which may lead to rinsing out of methanogenic sludge from the reactor.
Yet another disadvantage of known processes is that the elemental sulfur that is formed does not accumulate exclusively in the sulfur settling tank, but also forms deposits on walls of the tubing, vessels, sprayers, blowers, pumps, packing material, etc., which causes the need for regular cleaning of the equipment.
It is an object of the present invention to provide a process which obviates, at least partly, some or all of the above mentioned problems. The process of the invention thus aims to provide an improvement to anaerobic wastewater treatment with elemental sulfur recovery.
In accordance with the present invention, there is provided a process for removal of sulfur compounds from wastewater, comprising the steps of: a) converting in a first anaerobic suspended sludge reactor organic compounds present in the wastewater to acid compounds, forming an effluent comprising the acid compounds; b) converting the sulfur compounds to sulfide compounds in the first reactor, the remainder of the sulfur compounds being also comprised by said effluent; c) removing sulfide compounds in said first reactor with a stripping gas, forming a gaseous effluent; d) converting the acid compounds in said effluent to biogas in a second anaerobic reactor; and e) converting sulfur compounds in said effluent to sulfide compounds in the second anaerobic reactor, forming a second liquid effluent.
It will be understood that by converting the compounds in steps a), b), d) and e), and by stripping in step c) is meant that at least an essential part of the respective compounds present are converted, stripped or removed, the non-converted, non-stripped and non-removed compounds leaving the reactor in the effluent.
The organic compounds present in the wastewater are converted to acid compounds, in particular organic (carboxylic) acids, such as (lower) fatty acids. The conversion of organic compounds to acid compounds, as well as the conversion of sulfur compounds to sulfide is established using microorganisms known to the skilled person. For acidification of organic compounds bacterial species from the genera Clostridium, Ruminococcus, Propionibacterium, Selenomonas, Micromonospora, the family of the Lactobacteriaceae, and thermophilic clostridia can be used, as well as anaerobic sludge in which they are present. For the production of sulfide from sulfur compounds bacterial species from the genera Desulfovibrio, Desulfotomaculum, Desulfobacter, Desulfococcus, Desulfuromonas, Desulfonema, Desulfobulbus and for thermophilic applications Sulfolobus can be used, as well as sulfate reducing sludge or anaerobic sludge in which they are present.
The first (acidification) reactor typically works at 10-40xc2x0 C. (mesophilic range) or 40-80xc2x0 C. (thermophilic range), preferably 30-35xc2x0 C., pressures between 0.3 and 4 bara, mostly 1 bara and biomass concentrations between 0.1 and 10 g dry weight/dm3, mostly 3 g/dm3. The ratio between stripping gas flow rate and wastewater flow rate can be 3 to 100, but is typically 15.
The term xe2x80x98suspended sludge reactorxe2x80x99 is used in the present description and claims in its ordinary meaning and encompasses, as the skilled person knows, reactors in which the sludge is essentially free to move through the reactor, viz. reactors in which the sludge is essentially not bound to a static surface in the reactor. Such reactors do therefore not rely on measures to increase the surface area which may serve as a substrate for the microorganisms, such as packings, etc. The sludge in these type of reactors is kept in suspension by means of agitation, e.g. by mixing using an agitator, by recirculating the liquids or by bubbling gas through the liquids.
As a result of the production of acid compounds, the pH in the first reactor will be relatively low. The pH in the first reactor will depend on the type of wastewater to be treated, but is generally maintained at a value lower than 9, preferably lower than 8, most preferably between 6 and 7.5. Because of the low pH, the sulfide formed in the first reactor is relatively easily stripped. Moreover, only a relatively small amount of CO2 is formed. A substantial amount, typically about 50% or more, of the sulfur compounds is converted to sulfide, and 50-100% of the produced sulfide can be absorbed in an absorber. The non-absorbed sulfide is passed to the second reactor, as well as other sulfur compounds, if present.
Apart from acid compounds, the effluent from the first reactor may comprise other organic compounds, such as organic compounds that have not been converted in the first reactor or organic compounds that are intermediates from the reactions in the first reactor. Essentially all types of organic compounds may be converted to biogas in the second anaerobic (methanogenic) reactor. The conversion to biogas, as well as the conversion of remaining sulfur compounds in this second reactor is established using microorganisms known to the skilled person. The bacterial species that are typically employed for conversion of sulfur compounds were mentioned above as typical species for converting sulfur in the first reactor. For methane production bacterial species from the genera Methanococcus, Methanobacterium, Methanocarcina, Methanothrix, Methanophanus and Methanobrevibacter can be used. For thermophilic applications Methanobacterium thermoautotrophicum or species from Methanothermus can be used, as well as anaerobic sludge in which they are present The second (methanogenic) reactor typically works at 10-40xc2x0 C. (mesophilic range) or 40-80xc2x0 C. (thermophilic range), preferably 30-35xc2x0 C., pressures between 0.3 and 4 bara, mostly 1 bara and biomass concentrations between 1 and 50 g dry weight/dm3, mostly 30 g/dm3. The ratio between stripping gas flow rate and wastewater flow rate can be 3 to 100, but is typically 20.
The sulfide loaded stripping gas leaves the first reactor and is processed in an absorber unit. A suitable absorber unit is described in EP-A-0 766 650 as mentioned herein-above. In detail, H2S is absorbed from a gaseous stream by contacting the gas with a regenerable redox liquor, having a pH from about 4 to 7. Preferably, the liquor comprises a transition metal complex, such as an Fe(III) complex, or chelated iron, such as ethylenediamine tetra-acetic acid (EDTA). The metal complex is preferably used in a concentration of about 0.01 to about 0.1 M.
Alternatively, the absorption of sulfide may be carried out with an alkaline scrubber, in which the gas is contacted with an alkaline solution and the H2S is converted to alkalihydrosulfide. The hydrosulfide may be converted to alkalisulfide (e.g. Na2S), which may be reused in e.g. a tannery process. This type of H2S absorber is particularly preferred for tannery wastewater treatment processes according to the invention, since Na2S is used as a raw material in tanneries.
The low CO2 production in the first reactor is very favourable. Since only little CO2 is produced, the sulfide loaded stripping gas will be relatively low in CO2 content. As a result, the sulfide may be removed from the gas, using means such as a conventional alkaline scrubbing process. If CO2 were to be produced in substantial amounts, this would be removed by the alkaline solution as well. This would result in high alkali consumption in the absorber and in a net removal of CO2 from the first reactor, leading to increased pH values in this reactor. For this reason, the low production of CO2 is a considerable advantage of the present invention.
Preferably the process of the invention further comprises a step f) for removing the sulfide compounds from the second effluent (i.e. the liquid effluent from the second reactor) with a stripping system. Again, by removing in step f) is meant that at least an essential part of the sulfide present is removed, the non-removed sulfide leaving the reactor in the effluent. By removing a substantial amount of sulfide in the first reactor, removal of sulfur compounds in the form of sulfide can be carried out more easily in the second reactor, since the concentration of sulfide in the effluent of the first reactor is already significantly lower. Typically, about 50% of the sulfur present in the wastewater is removed. Moreover, the methanogenesis process in the second reactor will be less influenced by the presence of sulfide. The use of two separate anaerobic reactors is thus advantageous, since it is not required to return desulfidized water to the methanogenic reactor to decrease sulfide concentration. This prevents (1) a too high hydraulic loading rate in this reactor and as a consequence (2) risk of sludge wash out, as well as (3) lower methanogenic activities. This is in particular important for wastewater types which do not lead to granular sludge formation in methanogenic reactors, but to the formation of flocculant sludge, e.g. tannery wastewater.
Another important aspect of the present invention is that the sulfide is removed from the first reactor by feeding a stripping gas through the liquid in the reactor, e.g. by bubbling. This makes the use of an external stripper superfluous, thus saving on installation and operation costs. Also, by using a stripping gas, no H2S will accumulate in the headspace of the first reactor, which would occur when the liquid was fed to an external stripper.
In this respect, mention is made of U.S. Pat. No. 4,614,588, which describes a method for reducing the sulfur content of wastewater by employing two reactors. Sulfide is removed by exhausting the gas from the first reactor. To obtain sufficient separation of H2S gas (i.e. to produce sufficient H2S pressure in the head space of the reactor), it is required in the process of U.S. Pat. No. 4,614,588 that the pH is kept below 6. Such a requirement poses a strong limitation on the versatility of the removal process, since alkaline wastewater, e.g. tannery wastewater, cannot suitably be treated in such a process, since it would lead to a pH of higher than 6.
Another advantage of flowing stripping gas through the first reactor is that it enables a good mixing of the water, the anaerobic sludge flocs and the gas, whereby the sludge may be suspended in the water by the gas. This enables the use of the anaerobic contact process (ACP). This feature is specifically constitutes an advantage over the disclosure of U.S. Pat. No. 4,735,723, which describes a process for the removal of sulfur compounds from wastewater using two anaerobic reactors. According to this document, a fluidized bed reactor is preferred to carry out the acidification reaction, preferably combined with an external stripper. In fluidized bed reactors a high sludge age is employed. As a result of the high sludge age, microorganisms will develop that will convert organic acids to methane. As a result, the pH of the first reactor will increase, which is undesirable as was set out above. Sludge ages that are typical for fluidized bed reactors are generally too high for the process of the present invention. The reactor systems suggested in U.S. Pat. No. 4,735,723 separate all water and sludge in the reactor, as a result of which the sludge remains in the reactor for a period of time that is generally too high for the process of the present invention.
According to the present invention it is essential that the average sludge age is kept low, preferably below 5 days, more preferably from 1-4 days, most preferably at about 3 days.
This can be obtained by carrying out the separation of water and sludge in an external separator. This can be obtained by using the ACP process, which forms a preferred embodiment of the invention, especially for the treatment of alkaline wastewater.
In the ACP process wastewater is mixed with recycled sludge solids and then converted in a reactor sealed off from the entry of air. The contents of the reactor are completely mixed and the sludge is present in the form of suspended flocs. The flocs are kept in suspension by agitation. In the reactor the bacteria in the flocs biologically convert the compounds in the wastewater to other compounds. After the reaction, the mixed liquor, containing sludge flocs, is transported to a settling tank in which the flocs settle and the relatively clear supernatant is the effluent. At the bottom section of the settler a more concentrated suspension of flocs is formed. A large part of this suspension is recycled to the reactor, a smaller part is discharged. By settling and recycling, the formation of flocs by biomass in the system is promoted (by selection). Because of the completely mixed characteristics of the system, the sludge retention time (sludge age) can be controlled precisely by the discharge flow rate of the settled sludge. For example, if every day one-third of the total mass of sludge is removed from the system, the sludge age will be 3 days. Since acetoclastic methanogens (which convert acetic acid into methane) need sludge ages of more than 5 days (because of their high doubling time), they will not grow in a system with a sludge age of 3 days.
The ACP process is typically used for wastewater with COD concentrations of 1500-5000 mg/l and the hydraulic retention time is typically 2-10 hours. Although problems have been reported with separation of sludge and gas in methanogenic anaerobic contact processes, in the present non-methanogenic system a better separation is obtained, as the gas is not produced in the flocs but added in form of bubbles in the water phase.
Since the methanogenesis from fatty acids is minimized using the ACP process, an optimal accumulation of fatty acids and a corresponding low pH is obtained.
Other types of reactors known in the art are less or not suitable to obtain a suitable sludge age. For example, packed bed reactors, such as anaerobic filter or fixed film reactors, do not enable the control of sludge age, since in these reactors the biomass (from which the sludge is formed) is fixed to the packing.
In this respect, reference is made to a publication by E. Sxc3xa4rner (Wat. Sci. Tech., 22 (1990) 395-404), which describes a sulphur removal process that uses an anaerobic trickling filter (Antric filter as a pretreatment step. The pH in this step is kept low by applying a recirculation stream over the filter. The accumulation of organic acids leads to a pH lower than 6, the minimum pH for methanogenesis. However, such low pH cannot be guaranteed in case alkaline wastewater is treated. Than the high pH and the high sludge age will lead to conversion of organic acids into methane. In addition, even under low pH conditions, methanogenic activity can occur in the Antric filter; according to Sxc3xa4rner, the choice for biofilm processes results in considerable CH4 formation, since it is inevitable that at different biofilm depths, different environmental conditions are found, among which conditions that give rise to breakdown of organic acids, whereby CH4 is produced. In addition, breakdown of organic acids results in a rise of pH, which makes this process unsuitable for treatment of alkaline waste water.
Also fluidized bed reactors are less suitable or even unsuitable, since these type of reactors are characterized by a considerable spread in residence time of the biomass. When fluidized support particles, such as sand, need to be partly refreshed, a portion of support particles covered with sludge will remain in the reactor. As the biomass growth on the fresh sand particles will be much slower than growth on the particles already covered with biomass, a large spread in sludge age will develop. As a result a considerable amount of biomass with a very high sludge age will be present in the reactor, which makes the development of acetoclastic methanogens possible, which results in the consumption of acetic acid.
Carrying out the desulfurization process in two separate reactors according to the invention, does not affect significantly the installation costs of the total plant.
By carrying out the process in accordance with the invention, the problem of precipitation of carbonate salts in the stripper as used in known processes is overcome. As a result of the lower pH, a lower flowrate of the stripping gas can be employed as well as a lower gas/liquid exchanging surface, making the need for an external (packed) stripper, operated with gas as the continuous phase unnecessary.
The biogas that is produced in the second reactor, will contain sulfide as well, which has to be removed. This can be done by conventional means, such as by the method described in EP-A-0 766 650, which was described herein-above as a means to remove the sulfide from the product gas of the first reactor. By removing the sulfide from the product gas, clean biogas is obtained, which can be used for a variety of purposes, which will be apparent to the skilled person. The sulfide is converted in the absorber to elemental sulfur. The sulfur containing effluent of the absorber is fed through conventional means, such as a coagulator, a flocculator and/or a settling tank to produce a sulfur slurry, which also can be used for a variety of purposes, which will be apparent to the skilled person.
The liquid effluent of the second reactor may still contain a considerable amount of sulfide. These sulfide compounds can be removed from the liquid effluent, using a stripping column, which is fed with a stripping gas comprising CO2. The stripping gas is brought into contact with the liquid by any suitable means, such as spraying or trickling the liquid down and flowing the stripping gas in countercurrent. The sulfide loaded stripping gas is then fed to an absorber unit, which is similar to the one described above, to convert the sulfide to elemental sulfur. The remaining desulfidized gas is vented.
Stripping of H2S from liquids proceeds more easily, i.e. at higher rates and/or more favorable equilibrium conditions, at relatively low pH (pH about 6.5 to 7.5) and higher temperatures (about 30-40xc2x0 C., preferably at about 31-35, most preferably at about 33xc2x0 C., instead of about 25xc2x0 C.). For this reason it is preferred to use a combustion off-gas as the stripping gas in stripping the H2S from the liquid effluent of the second reactor. The CO2 in the combustion off-gas provides a lower pH to the liquid, which enhances H2S stripping and prevents the formation of carbonate deposits in the stripper, whereas the heat present in the off-gas provides a higher temperature for the stripping process.
The combustion gases can be an effluent from conventional combustion processes. Another advantage of this embodiment is that no extra measures have to be taken to obtain CO2 externally. It is preferred to use biogas produced with the process of the invention for producing the off-gases, since it can be burned on-site to produce these off-gases.
In addition, it was surprisingly found that, despite the lower solubility of H2S in Fe(III)chelate containing liquors, the higher temperatures of the sulfide loaded stripping gas, which results in an increased temperature in the absorber, the rate of H2S absorption is enhanced in the absorption step. Also the flocculation of sulfur is enhanced by higher temperatures, as will be discussed below.
In a further preferred embodiment of the invention, the removing of the sulfide in step f) comprises contacting the liquid effluent from the second reactor with an oxygen containing gas, preferably air. Thus the stripping of the liquid effluent from the second reactor is carried out using a gas mixture, which further comprises oxygen. The oxygen concentration will generally be up to 20 vol. %. Preferably the oxygen concentration is from 5-20 vol. %, most preferably about 14 vol. %. To this end air can be mixed with the stripping gas, such as the off-gas mentioned above. In the process described in EP-A-0 766 650 the reduction of Fe(III) to Fe(II) by sulfide (by which sulfur is produced) and the reoxidation of Fe(II) is carried out by separate processes, i.e., in separate columns. According to EP-A-0 766 650 the oxygen concentration has to be carefully controlled in order to prevent introduction of oxygen in the system. It is therefore surprising that according to the present process it is not required to operate the absorption of sulfide in the absence of oxygen. The oxygen which is introduced through the stripping column in the absorber can instead be used to regenerate the Fe(II) to Fe(III) (or another suitable redox system that is used) in the absorber. This makes the presence of a regenerator no longer required in this embodiment. Also means to control the oxygen concentration are no longer required, adding to the economic advantage of this embodiment.
A further preferred embodiment which uses oxygen to regenerate the redox liquor, comprises feeding the sulfide from the gaseous effluent from the second reactor and the sulfide from the liquid effluent from the second reactor to separate absorbers using the same recirculating redox liquor, in which the sulfide from said gaseous effluent from the second reactor is contacted with said redox liquor in co-current. The stream of biogas is essentially smaller than the gaseous effluent of the stripper used to treat the liquid effluent of the second reactor. Therefore the absorber treating the biogas can be smaller as well. However, the same absorption liquid may be used, provided that transfer of oxygen from freshly regenerated redox liquor to the biogas is prevented. This can be obtained by operating the absorber for the biogas such that the biogas and the regenerated absorption liquid flow in co-current. The oxygen present in the absorption liquid thus can react with the reduced metal (e.g. Fe(II) is oxidized to Fe(III)).
In another preferred embodiment, the liquid effluent of the second reactor is passed over a stripper only once. In conventional processes the sulfide containing effluent of a methanogenic reactor has to be recirculated over a stripper in order to obtain an acceptable decrease in sulfide concentration. This is also caused by the unfavorable chemical equilibrium as a result of the high pH employed. A disadvantage of recirculation of the aqueous effluent is that the higher hydraulic loading rate of the stripper requires a larger stripper. The second prerequisite for a smaller stripper is that no recirculation of stripper effluent over the methanogenic reactor is required, which bears the disadvantage of too high hydraulic loading rates and sludge wash out. According to the present invention, however, recirculation is not required, since a substantial amount of sulfide is already removed in the first reactor (up to about 50%), as a result of which, the methanogenesis in the second reactor is hardly adversely affected. By using a suitable stripping gas, such as CO2 as described above, an overall sulfur removal of more than 90% can be obtained with the process of the invention, using no recirculation in the stripper for the liquid effluent of the second reactor.
Another preferred embodiment of the process of the invention is any of the processes as described above, in which step f) comprises contacting the sulfide containing biogas effluent and/or a gaseous sulfide containing effluent obtained from stripping the liquid effluent of the second reactor with a redox liquor at a pH of 7-9, preferably at a pH of about 8.5. In the art a pH of 4-7 is said to be optimal for operating the absorption step with a redox liquor, in order to minimize absorption of CO2 from the sulfide containing gas. If net removal CO2 from the system takes place, it is required to replenish it from external sources in order to prevent a pH that would be too high for operating the anaerobic reactor and the stripping process successfully. It was found, however, that the absorption of sulfide is enhanced when a higher pH of the redox liquor is employed. When a pH of about 7-9, preferably of about 8.5 is used, both the rate of absorption of H2S and the rate of conversion to elemental sulfur are increased. When conventional phosphate buffers are used in this liquor, this will give rise to considerable CO2 losses. However, when bicarbonate buffers are used, preferably in concentration of 0.5-1 M, CO2 losses can be prevented even at relatively high gas phase CO2 concentrations expected (about 10%).
In another preferred embodiment, step f) comprises contacting the sulfide containing biogas effluent and/or a gaseous sulfide containing effluent obtained from stripping the liquid effluent of the second reactor with a redox liquor, by which sulfide is converted to sulfur, forming a sulfur comprising effluent, which is fed to a coagulation tank, a flocculation tank and a settler tank, thus forming a sulfur rich effluent. It was found that deposition of solid sulfur on equipment, such as tubing, vessel, gas blowers, pumps and packing material, can be prevented by removing the sulfur as quickly as possible from the redox liquor. To this end the redox liquid is first fed to the coagulation tank, in which it is stirred vigorously for about 1-3 minutes, giving rise to the formation of small sulfur crystals After the coagulation tank the liquid is fed to a flocculation tank in which the suspension is stirred mildly for 3-10 minutes, by which the crystals will form larger flocs. Flocculation can further be enhanced by the addition of conventional flocculation agents. The flocs formed can be removed easily by means of the settler tank to which the liquid is fed next. It was further found that coagulation of sulfur crystals proceeds more rapidly at elevated temperatures, which constitutes another advantage for the use of off-gases from combustion processes.
The process of the invention is particularly useful in treating wastewater with high COD concentrations ( greater than 2000 mg/dm3).

Removal of sulfur compounds from wastewater

Nederlandse Organisatie Voor Toegepast - Natuurwetenschappelijk Onderzoek Tno


A secure programable timer allows connections to the internet via a phone line or a network during a programmed specific time range. A timer is set to close the connection from a computer (or telephone) to the phone line or network during a programmed time span. The computer is not connected to the phone line or network outside of the programmed time span. A timer motor rotates a timer that has programmable devices that interact with a gear to close the connection between the computer and the phone/network line during the programmed time, and to open the connection at time not in the programmed time range. A cover is secured over the timer and connection to the phone/network line to prevent a bypass connection to the phone/network line.

The invention relates to a programmable timer and circuits connected thereto, and more particularly to a protected timer and connect-disconnect circuit for Telephone, Cable and Network Connections.
A computer may be connected to the internet through a dial-up telephone line, a cable connection or a network. When the connection is through a dial-up connection, the computer is connected through a telephone line. This line usually is connected by a telephone cord connected to an outlet in the wall and a connection to the computer through a modem. In this instant, the cord commonly uses RJ11 plugs. When the connection is made through a cable modem or network, the cord commonly uses RJ45 plugs. In either circumstance, the cord is commonly connected between the computer and a wall outlet that is wired in to the house telephone or network lines.
Children often have a telephone connection is their room, and tend to stay on the internet late into the night, or to make telephone calls after the time they should be asleep in bed. It is desirable to have control over the telephone and internet connection so that the use is not abused by extending it late into the night.
The invention is a secure programable timer for allowing connections to the internet via a phone line or a network during a programmed specific time range. A timer is set to close the connection from a computer to the phone line or network during a programmed time span. The computer, or a telephone, is not connected to the phone line or network outside of the programmed time span. A timer motor rotates a timer that has programmable devices that interact with a gear to close the connection between the computer and the phone/network line during the programmed time, and to open the connection at times not in the programmed time range. A cover is secured over the timer and connection to the phone/network line to prevent a bypass connection to the phone/network line.

Timer connect-disconnect for telephone, cable and network connections