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1.
Pharmaceutics ; 16(4)2024 Mar 27.
Article in English | MEDLINE | ID: mdl-38675132

ABSTRACT

NDH-4338 is a highly lipophilic prodrug comprising indomethacin and an acetylcholinesterase inhibitor. A design of experiments approach was used to synthesize, characterize, and evaluate the wound healing efficacy of optimized NDH-4338 nanosuspensions against nitrogen mustard-induced skin injury. Nanosuspensions were prepared by sonoprecipitation in the presence of a Vitamin E TPGS aqueous stabilizer solution. Critical processing parameters and material attributes were optimized to reduce particle size and determine the effect on dissolution rate and burn healing efficacy. The antisolvent/solvent ratio (A/S), dose concentration (DC), and drug/stabilizer ratio (D/S) were the critical sonoprecipitation factors that control particle size. These factors were subjected to a Box-Behnken design and response surface analysis, and model quality was assessed. Maximize desirability and simulation experiment optimization approaches were used to determine nanosuspension parameters with the smallest size and the lowest defect rate within the 10-50 nm specification limits. Optimized and unoptimized nanosuspensions were prepared and characterized. An established depilatory double-disc mouse model was used to evaluate the healing of nitrogen mustard-induced dermal injuries. Optimized nanosuspensions (A/S = 6.2, DC = 2% w/v, D/S = 2.8) achieved a particle size of 31.46 nm with a narrow size range (PDI = 0.110) and a reduced defect rate (42.2 to 6.1%). The optimized nanosuspensions were stable and re-dispersible, and they showed a ~45% increase in cumulative drug release and significant edema reduction in mice. Optimized NDH-4338 nanosuspensions were smaller with more uniform sizes that led to improved physical stability, faster dissolution, and enhanced burn healing efficacy compared to unoptimized nanosuspensions.

2.
Bioorg Chem ; 103: 104128, 2020 10.
Article in English | MEDLINE | ID: mdl-32745761

ABSTRACT

A set of 4-(R2-imino)-3-mercapto-5-(R1)-4H-1,2,4-triazoles derivatives were synthesized, characterized and evaluated for their ability to inhibit nitric oxide (NO) production in PAM212 mouse keratinocytes, which led to the discovery and the subsequent evaluation of their growth inhibitory cytotoxic potency toward that same mouse cell line together with a number of human cells lines (PC3, HT-29 and HeLa). Some limited SAR could be established for both NO production inhibition potency and growth inhibition cytotoxicity. Noticeably, the compounds designed to be nitrofurantoin mimics were the most potent anti-neoplastic agents.


Subject(s)
Antineoplastic Agents/pharmacology , Growth Inhibitors/pharmacology , Imines/pharmacology , Nitric Oxide Synthase Type II/antagonists & inhibitors , Triazoles/pharmacology , Animals , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Proliferation/drug effects , Cells, Cultured , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Growth Inhibitors/chemical synthesis , Growth Inhibitors/chemistry , Imines/chemical synthesis , Imines/chemistry , Mice , Molecular Structure , Nitric Oxide/antagonists & inhibitors , Nitric Oxide/biosynthesis , Nitric Oxide Synthase Type II/metabolism , Structure-Activity Relationship , Triazoles/chemical synthesis , Triazoles/chemistry
3.
Exp Mol Pathol ; 114: 104410, 2020 06.
Article in English | MEDLINE | ID: mdl-32113906

ABSTRACT

Nitrogen mustard (NM) is a highly reactive bifunctional alkylating agent that induces inflammation, edema and blistering in skin. An important mechanism mediating the action of NM and related mustards is oxidative stress. In these studies a modified murine patch-test model was used to analyze DNA damage and the antioxidant/stress response following NM exposure in isolated epidermis. NM (20 µmol) was applied to glass microfiber filters affixed to a shaved dorsal region of skin of CD-1 mice. NM caused structural damage to the stratum corneum as reflected by increases in transepidermal water loss and skin hydration. This was coordinate with edema, mast cell degranulation and epidermal hyperplasia. Within 3 h of NM exposure, a 4-fold increase in phosphorylated histone H2AX, a marker of DNA double-stranded breaks, and a 25-fold increase in phosphorylated p53, a DNA damage marker, were observed in the epidermis. This was associated with a 40% increase in 8-oxo-2'-deoxyguanosine modified DNA in the epidermis and a 4-fold increase in 4-hydroxynonenal modified epidermal proteins. At 12 h post NM, there was a 3-75 fold increase in epidermal expression of antioxidant/stress proteins including heme oxygenase-1, thioredoxin reductase, superoxide dismutase, glutathione reductase, heat shock protein 27 and cyclooxygenase 2. These data indicate that NM induces early oxidative epidermal injury in mouse skin leading to an antioxidant/stress response. Agents that enhance this response may be useful in mitigating mustard-induced skin injury.


Subject(s)
Antioxidants/metabolism , Epidermis/metabolism , Mechlorethamine/pharmacology , Stress, Physiological/genetics , Alkylating Agents/pharmacology , Alkylating Agents/toxicity , Animals , Apoptosis/drug effects , Cyclooxygenase 2/genetics , DNA Damage/drug effects , Epidermis/drug effects , Glutathione Reductase/genetics , HSP27 Heat-Shock Proteins/genetics , Heme Oxygenase-1/genetics , Humans , Inflammation/chemically induced , Inflammation/genetics , Inflammation/pathology , Mechlorethamine/toxicity , Mice , Oxidative Stress/drug effects , Phosphorylation/drug effects , Skin/drug effects , Skin/metabolism , Superoxide Dismutase/genetics , Thioredoxin-Disulfide Reductase/genetics
4.
Bioorg Chem ; 89: 103014, 2019 08.
Article in English | MEDLINE | ID: mdl-31170642

ABSTRACT

Seventy-one 7-oxycoumarins, 66 synthesized and 5 commercially sourced, were tested for their ability to inhibit growth in murine PAM212 keratinocytes. Forty-nine compounds from the library demonstrated light-induced lethality. None was toxic in the absence of UVA light. Structure-activity correlations indicate that the ability of the compounds to inhibit cell growth was dependent not only on their physiochemical characteristics, but also on their ability to absorb UVA light. Relative lipophilicity was an important factor as was electron density in the pyrone ring. Coumarins with electron withdrawing moieties - cyano and fluoro at C3 - were considerably less active while those with bromines or iodine at that location displayed enhanced activity. Coumarins that were found to inhibit keratinocyte growth were also tested for photo-induced DNA plasmid nicking. A concentration-dependent alteration in migration on neutral gels caused by nicking was observed.


Subject(s)
Coumarins/pharmacology , Keratinocytes/drug effects , Photosensitizing Agents/pharmacology , Animals , Cell Proliferation/drug effects , Cells, Cultured , Coumarins/chemical synthesis , Coumarins/chemistry , Dose-Response Relationship, Drug , Mice , Molecular Structure , Photochemical Processes , Photosensitizing Agents/chemical synthesis , Photosensitizing Agents/chemistry , Structure-Activity Relationship
5.
Bioorg Med Chem Lett ; 29(4): 619-622, 2019 02 15.
Article in English | MEDLINE | ID: mdl-30638875

ABSTRACT

Linear furocoumarins, also known as psoralens, are clinically useful photo-activated pharmaceuticals employed to address hyperproliferative skin diseases. Seven diverse cytotoxic pharmacophores have been synthetically attached to 8-methoxypsoralen via a 5-amino functionality. The resulting unique set of compounds was evaluated for dark and light toxicity against PAM212 keratinocytes in culture.


Subject(s)
Cell Survival/drug effects , Darkness , Light , Methoxsalen/pharmacology , Photosensitizing Agents/pharmacology , Cells, Cultured , Humans , Keratinocytes/drug effects , Keratinocytes/radiation effects , Methoxsalen/chemistry , Photosensitizing Agents/chemistry , Skin Diseases/pathology
6.
Photochem Photobiol ; 94(3): 577-582, 2018 05.
Article in English | MEDLINE | ID: mdl-29315592

ABSTRACT

Photosensitizers are used in the treatment of epidermal proliferation and differentiation disorders such as psoriasis and vitiligo. In these studies, a ring-expanded carbon homolog of the linear psoralen (furo[3,2-g]benzopyran-7-one) class of photosensitizers, 4,10-dimethyl-2H,8H-benzo[1,2-b:5,4-b']dipyran-2-one (NDH2476), was synthesized and analyzed for biological activity. Following activation by ultraviolet light (UVA, 320-400 nm), NDH2476 was found to be a potent inhibitor of keratinocyte growth (IC50  = 9 nm). Similar derivatives methylated in the pyran ring, or containing a saturated pyran ring structure, were markedly less active or inactive as photosensitizers. NDH2476 was found to intercalate and damage DNA following UVA light treatment as determined by plasmid DNA unwinding and nicking experiments. Taken together, these data demonstrate that an intact furan ring in psoralen photosensitizers is not required for keratinocyte growth inhibition or DNA damage. Our findings that low nanomolar concentrations of a benzopyranone derivative were active as a photosensitizer indicates that this or a structurally related compound may be useful in the treatment of skin diseases involving aberrant epidermal cell growth and differentiation.


Subject(s)
Keratinocytes/drug effects , Photosensitizing Agents/chemistry , Photosensitizing Agents/pharmacology , Pyranocoumarins/chemistry , Pyranocoumarins/pharmacology , Cell Proliferation/drug effects , DNA Damage , Humans , Keratinocytes/cytology , Ultraviolet Rays
7.
Heterocycl Lett ; 8(4): 729-736, 2018.
Article in English | MEDLINE | ID: mdl-33575202

ABSTRACT

The natural product 8-methoxypsoralen (methoxsalen or 8-MOP) in combination with long wavelength ultraviolet light (UVA, 320-400 nm), also referred to as PUVA therapy, is used for the treatment of cutaneous proliferative disorders including psoriasis, vitiligo and mycosis fungoides. The use of 8-MOP (3) is limited by its poor water solubility and there remains a need to develop more water-soluble psoralens to enhance bioavailability following oral administration of the drug. In the present studies a water-soluble dimethylaminoethyl ether analog of 8-MOP was synthesized and analyzed for biological activity. This analog, (8-[2-(N,N-dimethylamino)ethoxy]-psoralen hydrochloride (1) [or CAS name: 9-[2-(dimethylamino)ethoxy]-7H-furo[3,2-g][1]benzopyran-7-one, hydrochloride], was found to be significantly more active than 3 in keratinocyte growth inhibition assays (IC50 = 12 nM and 130 nM for 1 and 3, respectively). The partially reduced dihydro derivative of 1, 8-[2-(N,N-dimethylamino)ethoxy]-4',5'-dihydropsoralen hydrochloride (2) [or CAS name: 9-[2-(dimethylamino)ethoxy]-2,3-dihydro-7H-furo[3,2-g][1]benzopyran-7-one, hydrochloride] and the partially reduced 4',5'-dihydro-8-methoxypsoralen (4) lacking the water-solubilizing side-chain were significantly less active. As inhibitors of keratinocyte growth they ranked as IC50 = 13,000 nM and 70,000 nM for 2 and 4, respectively, indicating that an unsaturated furan ring in the psoralen was required for maximal activity. Compound (1) was found to readily intercalate and damage DNA following UVA light treatment as determined by plasmid DNA nicking and unwinding experiments in neutral and alkaline agarose gels. Taken together, these data demonstrate that a water-soluble dimethylaminoethyl ether psoralen targets DNA, is highly active as a photosensitizer, and may be useful in the treatment of skin diseases involving abnormal keratinocyte proliferation.

8.
Toxicol Lett ; 293: 77-81, 2018 Sep 01.
Article in English | MEDLINE | ID: mdl-29127031

ABSTRACT

Sulfur mustard (SM, bis(2-chloroethyl sulfide) is a potent vesicating agent known to cause skin inflammation, necrosis and blistering. Evidence suggests that inflammatory cells and mediators that they generate are important in the pathogenic responses to SM. In the present studies we investigated the role of mast cells in SM-induced skin injury using a murine vapor cup exposure model. Mast cells, identified by toluidine blue staining, were localized in the dermis, adjacent to dermal appendages and at the dermal/epidermal junction. In control mice, 48-61% of mast cells were degranulated. SM exposure (1.4g/m3 in air for 6min) resulted in increased numbers of degranulated mast cells 1-14days post-exposure. Treatment of mice topically with an indomethacin choline bioisostere containing prodrug linked by an aromatic ester-carbonate that targets cyclooxygenases (COX) enzymes and acetylcholinesterase (1% in an ointment) 1-14days after SM reduced skin inflammation and injury and enhanced tissue repair. This was associated with a decrease in mast cell degranulation from 90% to 49% 1-3days post SM, and from 84% to 44% 7-14days post SM. These data suggest that reduced inflammation and injury in response to the bifunctional indomethacin prodrug may be due, at least in part, to abrogating mast cell degranulation. The use of inhibitors of mast cell degranulation may be an effective strategy for mitigating skin injury induced by SM.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Cell Degranulation/drug effects , Chemical Warfare Agents/toxicity , Cholinergic Antagonists/pharmacology , Mast Cells/drug effects , Mustard Gas/toxicity , Prodrugs/pharmacology , Skin/cytology , Skin/drug effects , Animals , Choline/pharmacology , Cyclooxygenase Inhibitors/pharmacology , Dermatitis/drug therapy , Indomethacin/pharmacology , Male , Mice , Mice, Hairless , Wound Healing/drug effects
9.
Ann N Y Acad Sci ; 1378(1): 174-179, 2016 08.
Article in English | MEDLINE | ID: mdl-27505078

ABSTRACT

The molecular pathology of sulfur mustard injury is complex, with at least nine inflammation-related enzymes and receptors upregulated in the zone of the insult. A new approach wherein inhibitors of these targets have been linked by hydrolyzable bonds, either one to one or via separate preattachment to a carrier molecule, has been shown to significantly enhance the therapeutic response compared with the individual agents. This article reviews the published work of the authors in this drug development domain over the last 8 years.


Subject(s)
Anti-Inflammatory Agents/administration & dosage , Chemical Warfare Agents/toxicity , Drug Delivery Systems/methods , Mustard Gas/toxicity , Prodrugs/administration & dosage , Skin/drug effects , Animals , Anti-Inflammatory Agents/metabolism , Chemical Warfare Agents/metabolism , Cholinesterase Inhibitors/administration & dosage , Cholinesterase Inhibitors/metabolism , Cyclooxygenase Inhibitors/administration & dosage , Cyclooxygenase Inhibitors/metabolism , Drug Delivery Systems/trends , Drug Discovery/trends , Humans , Mustard Gas/metabolism , Prodrugs/metabolism , Skin/injuries , Skin/metabolism
10.
Exp Mol Pathol ; 100(3): 522-31, 2016 06.
Article in English | MEDLINE | ID: mdl-27189522

ABSTRACT

Nitrogen mustard (NM) is a bifunctional alkylating agent that is highly reactive in the skin causing extensive tissue damage and blistering. In the present studies, a modified cutaneous murine patch model was developed to characterize NM-induced injury and to evaluate the efficacy of an indomethacin pro-drug in mitigating toxicity. NM (20µmol) or vehicle control was applied onto 6mm glass microfiber filters affixed to the shaved dorsal skin of CD-1 mice for 6min. This resulted in absorption of approximately 4µmol of NM. NM caused localized skin damage within 1 d, progressing to an eschar within 2-3 d, followed by wound healing after 4-5 d. NM-induced injury was associated with increases in skin thickness, inflammatory cell infiltration, reduced numbers of sebocytes, basal keratinocyte double stranded DNA breaks, as measured by phospho-histone 2A.X expression, mast cell degranulation and increases in inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Wound healing was characterized by epidermal hyperplasia and marked increases in basal cells expressing proliferating cell nuclear antigen. A novel indomethacin-anticholinergic prodrug (4338) designed to target cyclooxygenases and acetylcholinesterase (AChE), was found to markedly suppress NM toxicity, decreasing wound thickness and eschar formation. The prodrug also inhibited mast cell degranulation, suppressed keratinocyte expression of iNOS and COX-2, as well as markers of epidermal proliferation. These findings indicate that a novel bifunctional pro-drug is effective in limiting NM mediated dermal injury. Moreover, our newly developed cutaneous patch model is a sensitive and reproducible method to assess the mechanism of action of countermeasures.


Subject(s)
Anti-Inflammatory Agents/pharmacology , Indomethacin/analogs & derivatives , Mechlorethamine/toxicity , Prodrugs/pharmacology , Skin/drug effects , Alkylating Agents/toxicity , Animals , Anti-Inflammatory Agents/chemistry , Cholinergic Antagonists/chemistry , Cholinergic Antagonists/pharmacology , Cyclooxygenase 2/metabolism , Cyclooxygenase Inhibitors/chemistry , Cyclooxygenase Inhibitors/pharmacology , DNA Damage , Female , Histones/metabolism , Immunohistochemistry , Indomethacin/chemistry , Indomethacin/pharmacology , Keratinocytes/drug effects , Keratinocytes/metabolism , Mice , Nitric Oxide Synthase Type II/metabolism , Prodrugs/chemistry , Proliferating Cell Nuclear Antigen/metabolism , Skin/injuries , Skin/pathology , Time Factors , Wound Healing/drug effects
11.
Invest Ophthalmol Vis Sci ; 57(4): 1687-98, 2016 Apr.
Article in English | MEDLINE | ID: mdl-27058125

ABSTRACT

PURPOSE: Sulfur mustard, nitrogen mustard (NM), and 2-chloroethyl ethyl sulfide all cause corneal injury with epithelial-stromal separation, differing only by degree. Injury can resolve in a few weeks or develop into chronic corneal problems. These vesicants induce microbullae at the epithelial-stromal junction, which is partially caused by cleavage of transmembranous hemidesmosomal collagen XVII, a component anchoring the epithelium to the stroma. ADAM17 is an enzyme involved in wound healing and is able to cleave collagen XVII. The activity of ADAM17 was inhibited in vesicant-exposed corneas by four different hydroxamates, to evaluate their therapeutic potential when applied 2 hours after exposure, thereby allowing ADAM17 to perform its early steps in wound healing. METHODS: Rabbit corneal organ cultures exposed to NM for 2 hours were washed, then incubated at 37°C for 22 hours, with or without one of the four hydroxamates (dose range, 0.3-100 nmol in 20 µL, applied four times). Corneas were analyzed by light and immunofluorescence microscopy, and ADAM17 activity assays. RESULTS: Nitrogen mustard-induced corneal injury showed significant activation of ADAM17 levels accompanying epithelial-stromal detachment. Corneas treated with hydroxamates starting 2 hours post exposure showed a dose-dependent ADAM17 activity inhibition up to concentrations of 3 nmol. Of the four hydroxamates, NDH4417 (N-octyl-N-hydroxy-2-[4-hydroxy-3-methoxyphenyl] acetamide) was most effective for inhibiting ADAM17 and retaining epithelial-stromal attachment. CONCLUSIONS: Mustard exposure leads to corneal epithelial sloughing caused, in part, by the activation of ADAM17 at the epithelial-stromal junction. Select hydroxamate compounds applied 2 hours after NM exposure mitigated epithelial-stromal separation.


Subject(s)
ADAM Proteins/metabolism , Corneal Diseases/metabolism , Epithelium, Corneal/metabolism , Mechlorethamine/toxicity , ADAM17 Protein , Animals , Blotting, Western , Cells, Cultured , Corneal Diseases/chemically induced , Corneal Diseases/pathology , Corneal Stroma/drug effects , Corneal Stroma/metabolism , Corneal Stroma/pathology , Epithelium, Corneal/drug effects , Epithelium, Corneal/pathology , Humans , Rabbits , Tomography, Optical Coherence , Tumor Necrosis Factor-alpha
12.
Toxicol Appl Pharmacol ; 303: 30-44, 2016 07 15.
Article in English | MEDLINE | ID: mdl-27125198

ABSTRACT

Vesicants including sulfur mustard (SM) and nitrogen mustard (NM) are bifunctional alkylating agents that cause skin inflammation, edema and blistering. This is associated with alterations in keratinocyte growth and differentiation. Endogenous cannabinoids, including N-arachidonoylethanolamine (anandamide, AEA) and 2-arachidonoyl glycerol (2-AG), are important in regulating inflammation, keratinocyte proliferation and wound healing. Their activity is mediated by binding to cannabinoid receptors 1 and 2 (CB1 and CB2), as well as peroxisome proliferator-activated receptor alpha (PPARα). Levels of endocannabinoids are regulated by fatty acid amide hydrolase (FAAH). We found that CB1, CB2, PPARα and FAAH were all constitutively expressed in mouse epidermis and dermal appendages. Topical administration of NM or SM, at concentrations that induce tissue injury, resulted in upregulation of FAAH, CB1, CB2 and PPARα, a response that persisted throughout the wound healing process. Inhibitors of FAAH including a novel class of vanillyl alcohol carbamates were found to be highly effective in suppressing vesicant-induced inflammation in mouse skin. Taken together, these data indicate that the endocannabinoid system is important in regulating skin homeostasis and that inhibitors of FAAH may be useful as medical countermeasures against vesicants.


Subject(s)
Alkylating Agents/toxicity , Chemical Warfare Agents/toxicity , Irritants/toxicity , Mechlorethamine/toxicity , Mustard Gas/toxicity , Skin/drug effects , Amidohydrolases/antagonists & inhibitors , Amidohydrolases/metabolism , Animals , Female , Male , Mice , Mice, Hairless , PPAR alpha/metabolism , Receptor, Cannabinoid, CB1/metabolism , Receptor, Cannabinoid, CB2/metabolism , Skin/metabolism
13.
Toxicol Sci ; 151(1): 150-9, 2016 05.
Article in English | MEDLINE | ID: mdl-26880746

ABSTRACT

Cytochrome P450 (CYP) enzymes mediate mixed-function oxidation reactions important in drug metabolism. The aromatic heterocyclic cation, diphenyleneiodonium (DPI), binds flavin in cytochrome P450 reductase and inhibits CYP-mediated activity. DPI also inhibits CYP by directly interacting with heme. Herein, we report that DPI effectively inhibits a number of CYP-related monooxygenase reactions including NADPH oxidase, a microsomal enzyme activity that generates hydrogen peroxide in the absence of metabolizing substrates. Inhibition of monooxygenase by DPI was time and concentration dependent with IC50's ranging from 0.06 to 1.9 µM. Higher (4.6-23.9 µM), but not lower (0.06-1.9 µM), concentrations of DPI inhibited electron flow via cytochrome P450 reductase, as measured by its ability to reduce cytochrome c and mediate quinone redox cycling. Similar results were observed with inducible nitric oxide synthase (iNOS), an enzyme containing a C-terminal reductase domain homologous to cytochrome P450 reductase that mediates reduction of cytochrome c, and an N-terminal heme-thiolate oxygenase domain mediating nitric oxide production. Significantly greater concentrations of DPI were required to inhibit cytochrome c reduction by iNOS (IC50 = 3.5 µM) than nitric oxide production (IC50 = 0.16 µM). Difference spectra of liver microsomes, recombinant CYPs, and iNOS demonstrated that DPI altered heme-carbon monoxide interactions. In the presence of NADPH, DPI treatment of microsomes and iNOS yielded a type II spectral shift. These data indicate that DPI interacts with both flavin and heme in CYPs and iNOS. Increased sensitivity for inhibition of CYP-mediated metabolism and nitric oxide production by iNOS indicates that DPI targets heme moieties within the enzymes.


Subject(s)
Cytochrome P-450 Enzyme Inhibitors/pharmacology , Cytochrome P-450 Enzyme System/metabolism , Heme/antagonists & inhibitors , Nitric Oxide Synthase Type II/antagonists & inhibitors , Onium Compounds/pharmacology , Animals , Dose-Response Relationship, Drug , Heme/metabolism , Humans , Mice , Microsomes, Liver/drug effects , Microsomes, Liver/enzymology , Nitric Oxide/metabolism , Nitric Oxide Synthase Type II/metabolism , Rats, Sprague-Dawley , Recombinant Proteins/metabolism , Time Factors
14.
Bioorg Med Chem Lett ; 25(23): 5609-12, 2015 Dec 01.
Article in English | MEDLINE | ID: mdl-26510670

ABSTRACT

Novel ethynylphenyl carbonates and carbamates containing carbon- and silicon-based choline mimics were synthesized from their respective phenol and aniline precursors and screened for anticholinesterase and anti-inflammatory activities. All molecules were micromolar inhibitors of acetylcholinesterase (AChE), with IC50s of 28-86 µM; the carbamates were two-fold more potent than the carbonates. Two of the most potent AChE inhibitors suppressed 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced inflammation by 40%. Furthermore, these molecules have physicochemical properties in the range of other CNS drugs. These molecules have the potential to treat inflammation; they could also dually target Alzheimer's disease through restoration of cholinergic balance and inflammation suppression.


Subject(s)
Acetylcholinesterase , Anti-Inflammatory Agents/chemical synthesis , Carbamates/chemical synthesis , Carbonates/chemical synthesis , Cholinesterase Inhibitors/chemical synthesis , Acetylcholinesterase/chemistry , Anti-Inflammatory Agents/chemistry , Anti-Inflammatory Agents/pharmacology , Carbamates/chemistry , Carbamates/pharmacology , Carbonates/chemistry , Carbonates/pharmacology , Cholinesterase Inhibitors/chemistry , Cholinesterase Inhibitors/pharmacology , Humans , Inhibitory Concentration 50 , Molecular Structure
15.
J Pharmacol Exp Ther ; 352(3): 529-40, 2015 Mar.
Article in English | MEDLINE | ID: mdl-25550200

ABSTRACT

Sepiapterin reductase (SPR) catalyzes the reduction of sepiapterin to dihydrobiopterin (BH2), the precursor for tetrahydrobiopterin (BH4), a cofactor critical for nitric oxide biosynthesis and alkylglycerol and aromatic amino acid metabolism. SPR also mediates chemical redox cycling, catalyzing one-electron reduction of redox-active chemicals, including quinones and bipyridinium herbicides (e.g., menadione, 9,10-phenanthrenequinone, and diquat); rapid reaction of the reduced radicals with molecular oxygen generates reactive oxygen species (ROS). Using recombinant human SPR, sulfonamide- and sulfonylurea-based sulfa drugs were found to be potent noncompetitive inhibitors of both sepiapterin reduction and redox cycling. The most potent inhibitors of sepiapterin reduction (IC50s = 31-180 nM) were sulfasalazine, sulfathiazole, sulfapyridine, sulfamethoxazole, and chlorpropamide. Higher concentrations of the sulfa drugs (IC50s = 0.37-19.4 µM) were required to inhibit redox cycling, presumably because of distinct mechanisms of sepiapterin reduction and redox cycling. In PC12 cells, which generate catecholamine and monoamine neurotransmitters via BH4-dependent amino acid hydroxylases, sulfa drugs inhibited both BH2/BH4 biosynthesis and redox cycling mediated by SPR. Inhibition of BH2/BH4 resulted in decreased production of dopamine and dopamine metabolites, 3,4-dihydroxyphenylacetic acid and homovanillic acid, and 5-hydroxytryptamine. Sulfathiazole (200 µM) markedly suppressed neurotransmitter production, an effect reversed by BH4. These data suggest that SPR and BH4-dependent enzymes, are "off-targets" of sulfa drugs, which may underlie their untoward effects. The ability of the sulfa drugs to inhibit redox cycling may ameliorate ROS-mediated toxicity generated by redox active drugs and chemicals, contributing to their anti-inflammatory activity.


Subject(s)
Alcohol Oxidoreductases/antagonists & inhibitors , Alcohol Oxidoreductases/metabolism , Pterins/antagonists & inhibitors , Pterins/metabolism , Sulfasalazine/pharmacology , Sulfathiazoles/pharmacology , Alcohol Oxidoreductases/chemistry , Animals , Humans , Mice , Oxidation-Reduction/drug effects , PC12 Cells , Protein Structure, Secondary , Pterins/chemistry , Rats , Sulfathiazole
16.
Mod Res Inflamm ; 3(2): 48-58, 2014 May.
Article in English | MEDLINE | ID: mdl-25360396

ABSTRACT

A series of Nω-nitro-Nω'-substituted guanidines has been prepared as potential inhibitors of the human Nitric Oxide Synthase (NOS) isoforms. The reported utility of aminoguanidine and nitroarginine in iNOS inhibition points to a potential similar utility for analogs of nitro-guanidine. The compound library was tested against the three isoforms of Nitric Oxide Synthase (eNOS, iNOS and nNOS). Several candidates showed excellent activity and good selectivity for nNOS. One particular compound even demonstrated good selectivity for iNOS. The potential usefulness of such selective inhibitors is discussed.

17.
Toxicol Appl Pharmacol ; 280(2): 236-44, 2014 Oct 15.
Article in English | MEDLINE | ID: mdl-25127551

ABSTRACT

Sulfur mustard (bis(2-chloroethyl) sulfide, SM) is a highly reactive bifunctional alkylating agent inducing edema, inflammation, and the formation of fluid-filled blisters in the skin. Medical countermeasures against SM-induced cutaneous injury have yet to be established. In the present studies, we tested a novel, bifunctional anti-inflammatory prodrug (NDH 4338) designed to target cyclooxygenase 2 (COX2), an enzyme that generates inflammatory eicosanoids, and acetylcholinesterase, an enzyme mediating activation of cholinergic inflammatory pathways in a model of SM-induced skin injury. Adult SKH-1 hairless male mice were exposed to SM using a dorsal skin vapor cup model. NDH 4338 was applied topically to the skin 24, 48, and 72 h post-SM exposure. After 96 h, SM was found to induce skin injury characterized by edema, epidermal hyperplasia, loss of the differentiation marker, keratin 10 (K10), upregulation of the skin wound marker keratin 6 (K6), disruption of the basement membrane anchoring protein laminin 322, and increased expression of epidermal COX2. NDH 4338 post-treatment reduced SM-induced dermal edema and enhanced skin re-epithelialization. This was associated with a reduction in COX2 expression, increased K10 expression in the suprabasal epidermis, and reduced expression of K6. NDH 4338 also restored basement membrane integrity, as evidenced by continuous expression of laminin 332 at the dermal-epidermal junction. Taken together, these data indicate that a bifunctional anti-inflammatory prodrug stimulates repair of SM induced skin injury and may be useful as a medical countermeasure.


Subject(s)
Anti-Inflammatory Agents, Non-Steroidal/therapeutic use , Cholinergic Antagonists/therapeutic use , Mustard Gas/toxicity , Skin Diseases/drug therapy , Animals , Cyclooxygenase 2 , Ki-67 Antigen/analysis , Male , Matrix Metalloproteinase 9 , Mice , Mice, Hairless , Skin/pathology , Skin Diseases/chemically induced , Skin Diseases/pathology , Wound Healing/drug effects
18.
J Pharm Sci ; 102(6): 2033-2043, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23471831

ABSTRACT

SRX246 is a potent, highly selective, orally bioavailable vasopressin 1a receptor antagonist that represents a novel mechanism of action for the treatment of mood disorders. The compound previously showed efficacy in animal models of mood disorders and excellent safety and tolerability in healthy volunteers in phase I clinical trials. In this study, SRX246 was further characterized in rats and dogs. In vitro determinations of permeability, protein binding, hepatocyte metabolism, and cytochrome P450 enzyme inhibition and in vivo assessments of pharmacokinetics were conducted. In parallel artificial membrane permeability assay (PAMPA) and PAMPA-blood-brain barrier models, SRX246 was comparable to highly permeable, orally active pharmaceuticals. SRX246 hydrochloride salt was 95.5 ± 1.7%, 95.9 ± 1.3%, and 98.6 ± 0.4% bound to rat, dog, and human serum proteins, respectively, and was stable in serum after a 4 h incubation at 37°C. P450 enzyme inhibition results showed a very low potential for drug-drug interactions. Metabolism in primary hepatocytes demonstrated that SRX246 was stable in humans and moderately metabolized in dogs and rats. Plasma pharmacokinetics findings showed a half-life (T½ ) of 2 and 6 h in rat and dog, respectively. Rat brain levels following a single oral dose were approximately 20% of plasma values with a T½ of 6 h. The observed profile for SRX246 supports further development.


Subject(s)
Antidiuretic Hormone Receptor Antagonists , Azetidines/metabolism , Azetidines/pharmacokinetics , Animals , Azetidines/blood , Blood Proteins/metabolism , Blood-Brain Barrier/metabolism , Cytochrome P-450 Enzyme Inhibitors , Cytochrome P-450 Enzyme System/metabolism , Dogs , Female , Hepatocytes/metabolism , Humans , Male , Mood Disorders/drug therapy , Protein Binding , Rats , Rats, Sprague-Dawley
19.
Angew Chem Int Ed Engl ; 51(44): 11126-30, 2012 Oct 29.
Article in English | MEDLINE | ID: mdl-23037915

ABSTRACT

A series of inhibitors of acetylcholinesterase (AChE) have been screened by back-scattering interferometry (BSI). Enzyme levels as low as 100 pM (22,000 molecules of AChE) can be detected. This method can be used to screen for mixed AChE inhibitors, agents that have shown high efficacy against Alzheimer's disease, by detecting dual-binding interactions. E = enzyme, I = inhibitor, S = substrate.


Subject(s)
Acetylcholinesterase/chemistry , Cholinesterase Inhibitors/chemistry , Acetylcholinesterase/metabolism , Animals , Cholinesterase Inhibitors/pharmacology , Electrophorus , Interferometry , Light , Molecular Structure
20.
Bioorg Med Chem ; 20(3): 1337-45, 2012 Feb 01.
Article in English | MEDLINE | ID: mdl-22249122

ABSTRACT

SRX246 is a potent, highly selective human vasopressin V1a antagonist that crosses the blood-brain barrier in rats. CNS penetration makes SRX246 an ideal candidate for potential radiolabeling and use in visualization and characterization of the role of the V1a receptor in multiple stress-related disorders. Before radiolabeling studies, cold reference analogs of SRX246 were prepared. This study describes the synthesis and in vitro screening for human V1a receptor binding and permeability of fluoro, iodo, and methyl reference compounds for SRX246 and the preparation of a tin precursor. For each compound, the potential utility of corresponding radiolabeled analogs for PET and SPECT imaging is discussed.


Subject(s)
Antidiuretic Hormone Receptor Antagonists , Azetidines/chemical synthesis , Azetidines/pharmacology , Positron-Emission Tomography/methods , Radioisotopes/chemistry , Radioisotopes/pharmacology , Tomography, Emission-Computed, Single-Photon/methods , Arginine Vasopressin/metabolism , Blood-Brain Barrier/metabolism , Cell Line , Humans , Ligands , Protein Binding , Receptors, Vasopressin/analysis
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