Photoreactive Drug Research Articles

Cross-linked, cyclodextrin-based nanosponges for curcumin delivery - Physicochemical characterization, drug release, stability and cytotoxicity

Curcumin (CUR) is a poorly water-soluble and photoreactive drug having potent anticancer activity. The purpose of the study is to fabricate cyclodextrin nanosponges, for the delivery of curcumin using two crosslinkers, diphenyl carbonate (DPC) and pyromellitic dianhydride (PMDA) and to study the influence on drug solubility, stability and cytotoxicity. Solubility studies were performed with assorted cyclodextrin to crosslinker ratio and with selected drug nanosponge stoichiometric complex. The drug-loaded nanosponges were characterized using DSC, FTIR, XRD and SEM. Pore size and surface topology of nanosponges confirmed the nanochannels available for the drug to get entrapped. In vitro drug release revealed 16 fold increase in dissolution by CUR-PMDA-CDNS compared to the pure drug against 5 fold increase by CUR-DPC-CDNS. The photostability of CUR was enhanced to 1.7 fold in PMDA crosslinked nanosponges. In vitro cytotoxicity study revealed increased toxicity of drug nanosponge complex to MCF-7 cells at a lower concentration. IC50 value of the drug (22.51 μg/ml) was reduced by 2.2 fold (10.44 μg/ml) by CUR-PMDA-CDNS as against 1.4 fold (15.92 μg/ml) decrease by CUR-DPC-CDNS. In conclusion, PMDA-CDNS was found to be a potential nanocarrier compared to DPC-CDNS for curcumin.

Broad-spectrum sunscreens prevent the secretion of proinflammatory cytokines in human keratinocytes exposed to ultraviolet A and phototoxic lomefloxacin

The combination of phototoxic drugs and ultraviolet (UV) radiation can trigger the release of proinflammatory cytokines. The present study measured the ability of sunscreens to prevent cytokine secretion in human keratinocytes following cotreatment of these cells with a known photoreactive drug and UVA. Keratinocytes were treated for 1 h with increasing concentrations of lomefloxacin (LOM) or norfloxacin (NOR), exposed to 15 J/cm2 UVA, and incubated for 24 h. NOR, owing to the absence of a fluorine atom in position 8, was non-phototoxic and used as a negative control. Cell viability and the release of 3 cytokines were assessed, namely interleukin-1alpha (IL-1alpha), interleukin-6 (IL-6), and tumour necrosis factor-alpha (TNF-alpha). The measurement of these cytokines may be a useful tool for detecting photoreactive compounds. To measure their ability to prevent cytokine secretion, various sunscreens were inserted between the UVA source and the cells. Treatment with NOR, NOR plus UVA, or LOM had no effect on the cells. LOM plus UVA, however, had an effect on cell viability and on cytokine secretion. IL-1alpha levels increased with LOM concentration. The release of TNF-alpha and IL-6 followed the same pattern at lower concentrations of LOM but peaked at 15 micromol/L and decreased at higher concentrations. Sunscreens protected the cells from the effects of LOM plus UVA, as cell viability and levels of cytokines remained the same as in the control cells. In conclusion, the application of broad-spectrum sunscreen by individuals exposed to UVA radiation may prevent phototoxic reactions initiated by drugs such as LOM.

Arginine482 to threonine mutation in the breast cancer resistance protein ABCG2 inhibits rhodamine 123 transport while increasing binding.

ABCG2 [also known as BCRP (breast cancer resistance protein) or MXR] is an ABC (ATP-binding cassette) protein shown to confer multidrug resistance. ABCG2 was initially identified in resistant breast carcinoma cells (MCF-7/AdrVp1000) selected with doxorubicin and verapamil. Later studies demonstrated the presence of a point mutation (Arg482 to Thr) in ABCG2 in MCF-7/AdrVp1000 cells. This mutation was shown to modulate the transport of Rh123 (rhodamine 123). In the present study, we have used a previously characterized photoreactive drug analogue of Rh123, IAARh123 (iodoaryl-azido-Rh123), to examine the effects of the Arg482Thr mutation on Rh123 binding and transport by ABCG2. Our results show that both wild-type (ABCG2R482) and mutant (ABCG2T482) ABCG2 bound directly to IAARh123. Surprisingly, however, wild-type ABCG2R482, which does not transport Rh123, was more intensely photolabelled than mutant ABCG2T482. In addition, inhibition of IAARh123 photolabelling using various drug substrates of ABCG2 revealed some differences between wild-type and mutant ABCG2. For example, a molar excess of mitoxantrone was more effective at inhibiting IAARh123 labelling of wild-type than of mutant ABCG2, while excess cisplatin, taxol and methotrexate showed significant inhibition of IAARh123 binding to both wild-type and mutant ABCG2. Taken together, the results of this study provide the first demonstration of the direct binding of drugs to ABCG2.

Drug binding domains of MRP1 (ABCC1) as revealed by photoaffinity labeling.

Drug resistance is a major impediment in the treatment of cancer patients receiving single or multiple drug treatment. Efforts to reverse drug resistance of tumor cells have not been successful. In recent years, considerable emphasis has been placed on understanding the underlying mechanisms that confer drug resistance. The expression of the multidrug resistance protein 1 (MRP1 or ABCC1) in cancer cells has been shown to confer resistance to diverse classes of anti-cancer drugs. MRP1 is a member of the ATP-binding cassette (ABC) family whose function, in tumor cells, is to reduce drug accumulation through energized drug efflux. To learn more about the functions of MRP1 in tumor drug resistance, knowledge of the protein binding characteristics and the location of its binding sites are essential. Photoaffinity labeling (PAL) has emerged as a leading technique that can rapidly shed light on a protein's drug binding characteristics and ultimately drug binding domains. Several MRP1-specific photoreactive probes have been developed. PAL of MRP1 was first demonstrated with the quinoline-based drug, IAAQ. Other studies showed that the high affinity endogenous substrate of MRP1, LTC(4), has intrinsic photoreactive properties and binds within both N- and C-terminal domains of MRP1. LTC(4) is conjugated to glutathione (GSH), a property common to several MRP1 substrates. In addition, several unconjugated drugs have been identified that interact with MRP1: [(3)H]VF-13,159, IAAQ, IACI and IAARh123. Mapping studies showed that IACI and IAARh123 bind two sites within transmembrane (TM) regions 10-11 and 16-17 of MRP1. Interestingly, the GSH-dependent PAL of [(125)I]azidoAG-A and [(125)I]LY475776 occurs within, or proximal to TM 16-17. The PAL with several analogs of GSH, IAAGSH and azidophenacyl-[(35)S]GSH found to interact specifically with MRP1 within TM 10-11 and TM 16-17 in addition to binding two cytoplasmic regions in MRP1, L0 and L1. This review focuses on the use of PAL for studying MRP1 interactions with various drugs and cell metabolites. Furthermore, knowledge of MRP1 drug binding domains, as identified by PAL with various photoreactive drug analogs, provides an important first step towards more detailed analyses of MRP1 binding domains.

Photoaffinity labeling under non-energized conditions of a specific drug-binding site of the ABC multidrug transporter LmrA from Lactococcus lactis

The Lactococcus lactis multidrug resistance ABC transporter protein LmrA has been shown to confer resistance to structurally and functionally diverse antibiotics and anti-cancer drugs. Using a previously characterized photoreactive drug analogue of Rhodamine 123 (iodo-aryl azido-Rhodamine 123 or IAARh123), direct and specific photoaffinity labeling of LmrA in enriched membrane vesicles could be achieved under non-energized conditions. This photoaffinity labeling of LmrA occurs at a physiologically relevant site as it was inhibited by molar excess of ethidium bromide>Rhodamine 6G>vinblastine>doxorubicin>MK571 (a quinoline-based drug) while colchicine had no effect. The MDR-reversing agents PSC 833 and cyclosporin A were similarly effective in inhibiting IAARh123 photolabeling of LmrA and P-glycoprotein. In-gel digestion with Staphyloccocus aureus V8 protease of IAARh123-photolabeled LmrA revealed several IAARh123 labeled polypeptides, in addition to a 6.8kDa polypeptide that comprises the last two transmembrane domains of LmrA.

The multidrug resistance protein ABCC1 drug-binding domains show selective sensitivity to mild detergents

The multidrug resistance protein (ABCC1 or MRP1) causes resistance to multiple drugs through reduced drug accumulation. We have previously demonstrated direct interaction between MRP1 and unmodified drugs using photoreactive drug analogues. In this study, we describe the use of [ 125 I]iodoaryl azido-rhodamine123 (IAARh123)—a photoactive drug analogue of rhodamine 123, to study the effects of mild detergents and denaturing agents on MRP1-drug binding in membrane vesicles prepared from HeLa cells transfected with the MRP1 cDNA. Our results show that the zwitterionic detergent CHAPS and a nonionic detergent Brij35 inhibited the photolabeling of MRP1 with IAARh123. Sodium deoxycholate (SDC) and octyl-β-glucoside (OG), structurally similar to CHAPS and Brij35 and disrupting the lipid bilayer, showed a modest increase of MRP1 photolabeling with IAARh123. Proteolytic digestion of IAARh123 photolabeled MRP1 labeled in the presence or absence of various detergents (CHAPS, SDC, or OG) revealed identical photolabeled peptides. Consistent with the drug-binding results, non-toxic concentrations of CHAPS and Brij35 reversed vincristine and etoposide (VP16) toxicity in MRP1 expressing cells. Taken together, the results of this study show that MRP1–drug interaction can occur outside the lipid bilayer environment. However, this interaction is inhibited with certain mild detergents.

Major Photoaffinity Drug Binding Sites in Multidrug Resistance Protein 1 (MRP1) Are within Transmembrane Domains 10–11 and 16–17

MRP1 is an ABC (or ATP binding cassette) membrane transport protein shown to confer resistance to structurally dissimilar drugs. Studies of MRP1 topology suggested the presence of a hydrophobic N-domain with five potential membrane-spanning domains linked to an MDR1-like core (MSD1-NBD1-L1-MSD2-NBD2) by an intracellular linker domain (L0). MRP1-mediated multidrug resistance is thought to be due to enhanced drug efflux. However, little is known about MRP1-drug interaction and its drug binding site(s). We previously developed several photoreactive probes to study MRP1-drug interactions. In this report, we have used eight MRP1-HA variants that were modified to have hemagglutinin A (HA) epitopes inserted at different sites in MRP1 sequence. Exhaustive in-gel digestion of all IAARh123 photoaffinity-labeled MRP1-HA variants revealed the same profile of photolabeled peptides as seen for wild type MRP1. Photolabeling of the different MRP1-HA variants followed by digestion with increasing concentrations of trypsin or Staphylococcus aureus V8 protease (1:800 to 1:5 w/w) and immunoprecipitation with anti-HA mAb identified two small photolabeled peptides ( approximately 6-7 kDa) from MRP1-HA(574) and MRP1-HA(1222). Based on the location of the HA epitopes in the latter variants together with molecular masses of the two peptides, the photolabeled amino acid residues were localized to MRP1 sequences encoding transmembranes 10 and 11 of MSD1 (Ser(542)-Arg(593)) and transmembranes 16 and 17 of MSD2 (Cys(1205)-Glu(1253)). Interestingly, the same sequences in MRP1 were also photolabeled with a structurally different photoreactive drug, IACI, confirming the significance of transmembranes 10, 11, 16 and 17 in MRP1 drug binding. Taken together, the results in this study provide the first delineation of the drug binding site(s) of MRP1. Furthermore, our findings suggest the presence of common drug binding site(s) for structurally dissimilar drugs.

Reversal of MRP-mediated doxorubicin resistance with quinoline-based drugs

The overexpression of P-glycoprotein (P-gp) and the multidrug resistance-associated protein (MRP) have been shown to confer broad drug resistance in tumor cells. We have demonstrated previously direct binding between MRP and a quinoline-based photoreactive drug (iodo-azido-amino quinoline, IAAQ) (Vezmar et al., Biochem Biophys Res Commun 241: 104–111, 1997). In this report, we show the reversal of multidrug resistance in two MRP-overexpressing cell lines, HL60/AR and H69/AR, with four quinoline-based drugs. Non-toxic concentrations (5–20 μM) of chloroquine, quinine, quinidine, and primaquine potentiated the toxicity of doxorubicin in a concentration-dependent manner. These quinoline-based drugs showed a 5- to 10-fold decrease in the ic 50 of doxorubicin in H69/AR and HL60/AR cells. Primaquine was the most active, with modulation ratios of 10- and 5-fold versus 8- and 3-fold with MK-571 for H69/AR and HL60/AR, respectively. Moreover, using IAAQ, we showed that molar excesses of chloroquine, quinine, quinidine, and MK-571 inhibit the photoaffinity labeling of MRP. Primaquine and vinblastine showed lesser inhibition of MRP photoaffinity labeling by IAAQ. Taken together, the results of this study demonstrated the reversal of doxorubicin resistance with several quinoline-based drugs. Moreover, these drugs have been shown to reverse P-gp-mediated MDR and are clinically well tolerated.

Tin ethyl etiopurpurin—induced photodynamic therapy for the treatment of human immunodeficiency virus—associated kaposi's sarcoma

Current local treatments for cutaneous human immunodeficiency virus—associated Kaposi's sarcoma lesions can be less than ideal. Photodynamic therapy (PDT) consisting of a photoreactive drug and a laser light source has been used to treat a variety of neoplasms. This paper presents the case report of a 36-year-old man with multiple Kaposi's sarcoma lesions. Using the photosensitizer tin ethyl etiopurpurin and red light activation 24 hours postinfusion, we achieved excellent subjective and objective response on follow-up examinations to 20 weeks posttreatment. Photodynamic therapy is an outpatient procedure that is well tolerated and highly effective for the treatment of Kaposi's sarcoma lesions. It offers the distinct benefit of effective destruction of multiple lesions in a single session without severe morbidity. PDT should be considered as the first-line treatment of Kaposi's sarcoma, but it also should undergo large clinical trials to fully assess its capabilities

Evaluation and routine application of the novel restricted-access precolumn packing material Alkyl-Diol Silica: coupled-column high-performance liquid chromatographic analysis of the photoreactive drug 8-methoxypsoralen in plasma.

A fully automated coupled-column HPLC method for on-line sample processing and determination of the photoreactive drug 8-methoxypsoralen (8-MOP) in plasma has been developed. The method is based on the novel internal-surface reversed-phase precolumn packing materials Alkyl-Diol Silica (ADS). This new family of restricted-access materials has a hydrophilic, electroneutral outer particle surface and a hydrophobic internal pore surface. The supports tolerate the direct and repetitive injection of proteinaceous fluids such as plasma and allow a classical C18-, C8- or C4-reversed-phase partitioning at the internal (pore) surface. The total protein load, i.e. the lifetime of the precolumn used in this study (C8-Alkyl-Diol Silica, 25 microns, 25 x 4 mm I.D.), exceeds more than 100 ml of plasma. 8-MOP was detected by its native fluorescence (excitation 312 nm, emission 540 nm). Validation of the method revealed a quantitative and matrix-independent recovery (99.5-101.3% measured at five concentrations between 21.3 and 625.2 ng of 8-MOP per milliliter of plasma), linearity over a wide range of 8-MOP concentrations (1.2-3070 ng of 8-MOP/ml, r = 0.999), low limits of detection (0.39 ng of 8-MOP/ml) and quantitation (0.79 ng of 8-MOP/ml) and a high between-run (C.V. 1.47%, n = 10) and within-run (C.V. 1.33%, n = 10) reproducibility. This paper introduces coupled-column HPLC as a suitable method for on-site analysis of drug plasma profiles (bedside-monitoring).

A total delivery system of genetically engineered drugs or cells for diseased vessels. Concept, materials, and fabricated prototype device.

The development of a percutaneous procedure using a catheterized system for diseased vessels has been increasingly in demand in conjunction with gene therapy using genetically engineered drugs (antisense) and cells. The authors' strategic concept realizes revascularization at narrowed, diseased sites and delivery of drugs or cells into the diseased tissues or targeted cells. An inflatable, drug-releasing double balloon is installed at the tip of a catheter. The outer balloon, fabricated with micropores (diameters of 20 and 30 mm) by an excimer laser ablation technique, releases a viscous solution containing a photoreactive polymer and drug or cells on inflation of the inner balloon. A photoresponsive water-soluble polymer, molecularly designed for its ability to achieve prolonged local residency of antisense DNA at the tissue level and enhanced transmembrane transport at the cellular level, is premixed with antisense oligonucleotide drug. On light irradiation, the nonionic polymer is reversibly converted to a positively charged polymer that can be complexed with highly negatively charged antisense DNA (c-myb), which may enhance the transmembrane delivery of antisense. On cessation of irradiation, the complex slowly dissociates to function intracellularly as an antisense drug, resulting in inhibition of cell proliferation. Thus, our integrated, dual-function balloon system may contribute to mechanical dilatation gene therapies at diseased vessels.