Ginkgo biloba extract attenuates cisplatin-induced renal interstitial fibrosis by inhibiting the activation of renal fibroblasts through down-regulating the HIF-1α/STAT3/IL-6 pathway in renal tubular epithelial cells.

BACKGROUND: Activation of renal fibroblasts into myofibroblasts plays an important role in promoting renal interstitial fibrosis (RIF). Ginkgo biloba extract (EGb) can alleviate RIF induced by cisplatin (CDDP). PURPOSE: To elucidate the effect of EGb treatment on cisplatin-induced RIF and reveal its potential mechanism. METHODS: The two main active components in EGb were determined by high-performance liquid chromatography (HPLC) analysis. Rats were induced by CDDP and then treated with EGb, 2ME2 (HIF-1α inhibitor) or amifostine. After HK-2 cells and HIF-1α siRNA HK-2 cells were treated with CDDP, EGb or amifostine, the conditioned medium from each group was cultured with NRK-49F cells. The renal function of rats was detected. The renal damage and fibrosis were evaluated by H&E and Masson trichrome staining. The IL-6 content in the cell medium was detected by ELISA. The expression levels of indicators related to renal fibrosis and signaling pathway were examined by western blotting and qRT-PCR. RESULTS: HPLC analysis showed that the contents of quercetin and kaempferol in EGb were 36.0 µg/ml and 45.7 µg/ml, respectively. In vivo, EGb and 2ME2 alleviated renal damage and fibrosis, as well as significantly decreased the levels of α-SMA, HIF-1α, STAT3 and IL-6 in rat tissues induced by CDDP. In vitro, the levels of HIF-1α, STAT3 and IL-6 were significantly increased in HK-2 cells and HIF-1α siRNA HK-2 cells induced by CDDP. Notably, HIF-1α siRNA significantly decreased the levels of HIF-1α, STAT3 and IL-6 in HK-2 cells, as well as the IL-6 level in medium from HK-2 cells. Additionally, the α-SMA level in NRK-49F cells was significantly increased after being cultured with conditioned medium from HK-2 cells or HIF-1α siRNA HK-2 cells exposed to CDDP. Furthermore, exogenous IL-6 increased the α-SMA level in NRK-49F cells. Importantly, the expression levels of the above-mentioned indicators were significantly decreased after the HK-2 cells and HIF-1α siRNA HK-2 cells were treated with EGb. CONCLUSION: This study revealed that EGb improves CDDP-induced RIF, and the mechanism may be related to its inhibition of the renal fibroblast activation by down-regulating the HIF-1α/STAT3/IL-6 pathway in renal tubular epithelial cells.

Enteral Activation of WR-2721 Mediates Radioprotection and Improved Survival from Lethal Fractionated Radiation.

Unresectable pancreatic cancer is almost universally lethal because chemotherapy and radiation cannot completely stop the growth of the cancer. The major problem with using radiation to approximate surgery in unresectable disease is that the radiation dose required to ablate pancreatic cancer exceeds the tolerance of the nearby duodenum. WR-2721, also known as amifostine, is a well-known radioprotector, but has significant clinical toxicities when given systemically. WR-2721 is a prodrug and is converted to its active metabolite, WR-1065, by alkaline phosphatases in normal tissues. The small intestine is highly enriched in these activating enzymes, and thus we reasoned that oral administration of WR-2721 just before radiation would result in localized production of the radioprotective WR-1065 in the small intestine, providing protective benefits without the significant systemic side effects. Here, we show that oral WR-2721 is as effective as intraperitoneal WR-2721 in promoting survival of intestinal crypt clonogens after morbid irradiation. Furthermore, oral WR-2721 confers full radioprotection and survival after lethal upper abdominal irradiation of 12.5 Gy × 5 fractions (total of 62.5 Gy, EQD2 = 140.6 Gy). This radioprotection enables ablative radiation therapy in a mouse model of pancreatic cancer and nearly triples the median survival compared to controls. We find that the efficacy of oral WR-2721 stems from its selective accumulation in the intestine, but not in tumors or other normal tissues, as determined by in vivo mass spectrometry analysis. Thus, we demonstrate that oral WR-2721 is a well-tolerated, and quantitatively selective, radioprotector of the intestinal tract that is capable of enabling clinically relevant ablative doses of radiation to the upper abdomen without unacceptable gastrointestinal toxicity.

Possible association of radioprotective and chemoprotective aminophosphorothioate drug activity with polyamine oxidase susceptibility.

The aminophosphorothioate drug S-2-(3-aminopropylamino)ethylphosphorothioate (WR-2721) is both radioprotective and chemoprotective, with potential clinical use in cancer therapies. These distinct properties may be associated with its catabolism by polyamine oxidase (EC 1.4.3.4), which is found in varying amounts in different body tissues. Unfortunately, the metabolite is probably a cytotoxic aldehyde, but it should be detoxified in vivo following adduction with tissue sulfhydryls. Whether conversion and adduction benefit or hinder pharmacologic activity is not known, inasmuch as aldehydes may reduce oxygen-dependent free radicals generated by irradiation of tissues. S-2-(3-aminopropylamino)propylphosphorothioate was similarly a substrate for polyamine oxidase, and the product was cytotoxic. Other aminophosphorothioates [S-2-(4-aminobutaneamino)ethylphosphorothioate, S-2-(5-aminopentylamino)ethylphosphorothioate, and S-2-(4-aminobutaneamino)propylphosphorothioate] were poor substrates and less radioprotective.

Uptake of WR-2721 derivatives by cells in culture: identification of the transported form of the drug.

When V79-171 cells are incubated in medium to which WR-1065 has been added the cells accumulate WR-1065 and disulfides of WR-1065 (WRSS) in a ratio of about 10:1. Analysis of the culture medium showed that it contained primarily WR-1065 but that significant levels of the symmetrical disulfide WR-33278 and of the mixed disulfide of WR-1065 with cysteine were also present. Since incubation of cells with either of the latter disulfides did not lead to uptake it was concluded that WR-1065 is the form of the drug taken up. The uptake rate on a per cell basis was shown to be independent of cell density, to be first order in the WR-1065 concentration in the incubation medium, to increase as [H+]-1.2 at medium pH values from pH 6.8 to 8.0, and to have a Q10 value (rate increase per 10 degrees C temperature increase) of 2.9 +/- 0.3 between 2 and 37 degrees C. Rates of WR-1065 uptake measured for HeLa, HT29/SP-1d, Me-180-VCII, Ovary 2008, and WI-38 cell lines were found to be similar to that measured for V79-171 cells. The results are consistent with uptake by nonmediated, passive diffusion of the uncharged form of WR-1065 across the plasma membrane but uptake mediated by a membrane transport system could not be rigorously excluded. Based upon these results and earlier findings it is postulated that the lower drug uptake seen in tumors as compared with normal tissues in animals treated with WR-2721 results from a combination of (a) slower conversion of WR-2721 to WR-1065 in tumors as a consequence of the lower inherent level of alkaline phosphatase and lower pH in tumors and (b) a decreased uptake rate of the WR-1065 present in tumors as a consequence of their lower pH.

Measurement of tissue oxidation-reduction state with carbon-13 nuclear magnetic resonance spectroscopy.

The oxidation state of tissues influences their response to cancer therapy. We have devised a novel approach to the measurement of thiol redox which is based on the relative nuclear magnetic resonance signal intensity from carbon-13 adjacent to sulfur in metabolites of the redox-sensitive phosphorothioate drug, S-2-(3-methylaminopropylamino)ethylphosphorothioic acid (WR3689). Incubation of WR3689 metabolites under oxidizing conditions results in quantifiable changes in the 13C nuclear magnetic resonance spectrum stoichiometrically related to the degree of oxidation in mouse liver homogenate in vitro. Drug oxidation is competitive with the oxidation of tissue-derived thiol groups under these conditions. Noninvasive measurement of redox state may assist in designing more effective strategies for altering normal and malignant tissue response to cancer therapy.

Active versus passive absorption kinetics as the basis for selective protection of normal tissues by S-2-(3-aminopropylamino)-ethylphosphorothioic acid.

Through an in vivo and in vitro analysis of the absorption kinetics of S-2-(3-aminopropylamino)ethylphosphorothioic acid (WR-2721) in the normal tissues and solid tumors of mice, rats, and rabbits, it has been demonstrated that normal tissues actively concentrate WR-2721 against a concentration gradient, whereas solid tumors passively absorb it, or, if active concentration of WR-2721 is present in tumors, it operates at a far reduced rate relative to normal tissues. These observations can account for the ability of WR-2721 to selectively protect normal tissues against both radiation and alkylating agent injury.

Determination of WR-1065 in human blood by high-performance liquid chromatography following fluorescent derivatization by a maleimide reagent ThioGlo3.

In order to improve the sensitivity and stability of human blood samples containing WR-1065 (i.e., active metabolite of the cytoprotective agent amifostine), a high-performance liquid chromatographic method was developed and validated using fluorescent derivatization with ThioGlo3. Using a sample volume of only 100 microl, the method was specific, sensitive (limit of quantitation=10 nM in deproteinized blood or 20 nM in whole blood), accurate (error < or = 3.2%) and reproducible (CV < or = 8.7%). In addition, the stability of WR-1065 in deproteinized and derivatized blood samples was assured for at least four weeks at -20 degrees C. This method should be particularly valuable in translating the kinetic-dynamic relationship of WR-1065 in preclinical models to that in cancer patients.

WR-1065, an active metabolite of the cytoprotector amifostine, affects phosphorylation of topoisomerase II alpha leading to changes in enzyme activity and cell cycle progression in CHO AA8 cells.

The effects of WR-1065 (2-((aminopropyl)amino)ethanethiol) on cell cycle progression, topoisomerase (topo) II alpha activity, and topo II alpha phosphorylation in Chinese hamster ovary (CHO) cells have been investigated. Exposure of CHO cells to 0.4 microM of WR-1065 for 30 min did not effect cell cycle progression nor topo II alpha activity and phosphorylation status. However, concentrations ranging from 4 microM to 4 mM were equally effective in significantly altering these three end points. Cell cycle progression was analysed by flow cytometry. Following a 30 min exposure to this range of concentrations, cells redistributed throughout the cell cycle with the most prominent changes being an accumulation of cells in G2. Topo II alpha activity was measured using a kinetoplast DNA (kDNA) decatenation assay. Enzyme activity was reduced by 50% relative to control levels throughout the 4 microM to 4 mM dose range tested. Likewise, topo II alpha phosphorylation levels, analysed using an immunoprecipitation assay and an antibody specific to the 170 kDa band of topo II, decreased between 42% to 48% of control levels. Inhibition of topo II alpha activity in cells exposed to WR-1065 is consistent with the associated observation of WR-1065 mediated cell cycle progression delay and build-up of cells in the G2 phase of the cell cycle.

Radioprotectants: pharmacology and clinical applications of amifostine.

Amifostine (Ethyol, ALZA Pharmaceuticals, Palo Alto, CA/US Bioscience, West Conshohocken, PA) is a phosphorylated cysteamine derivative that was originally developed by the US Army Walter Reed Institute (Washington, DC) as a radioprotectant. Amifostine, a prodrug, is metabolized by the enzyme alkaline phosphatase to an active sulfhydryl compound (WR-1065) capable of scavenging radiation-generated free radicals and preventing cell damage. The disulfides of WR-1065 are structurally analogous to endogenous polyamines, which can bind to DNA molecules and stabilize them in a compact form less vulnerable to damage by cytotoxic agents. Preclinical and clinical studies show that amifostine is a selective radioprotectant that reduces both early and late radiation-induced toxicities to normal tissues while leaving tumor cells exposed to the cytotoxic effects of radiation. Preclinical data indicate that amifostine could reduce the risk of secondary cancers caused by radiation and certain forms of chemotherapy.

The preclinical basis for broad-spectrum selective cytoprotection of normal tissues from cytotoxic therapies by amifostine (Ethyol).

Administered prior to cytotoxic chemotherapy or radiation, the aminothiol amifostine provides broad-spectrum cytoprotection of various normal tissues without attenuating antitumour response. The basis for the selectivity of action resides in the anabolism of amifostine at the normal tissue site by membrane-bound alkaline phosphatase. Dephosphorylation to the free thiol, WR-1065, is followed by rapid uptake into normal tissues by a carrier mediated, facilitated diffusion process; in contrast, uptake into tumour tissue is slow to negligible. Preclinical studies have shown that pretreatment with amifostine provides protection of normal tissues from the cytotoxic effects of alkylating agents, organoplatinums, anthracyclines, taxanes and radiation. Normal tissues protected include bone marrow, kidney, neural tissues, the heart, intestinal crypt cells and pulmonary tissues. Additionally, the mutagenic and carcinogenic effects of these modalities are also attenuated. With respect to bone marrow, preclinical studies have shown significant protection of progenitor cells that give rise to the red and white cells and platelets. Comparative in vitro and in vivo studies using murine and human tumour xenografts show no decrease of antitumour effects of these same therapies despite the protection of normal organs. The unique preclinical profile of amifostine serves as a model for the clinical development programme for this important new broad-spectrum cytoprotective agent.

Amifostine: the preclinical basis for broad-spectrum selective cytoprotection of normal tissues from cytotoxic therapies.

Administered prior to cytotoxic chemotherapy or radiation, the aminothiol amifostine provides broad-spectrum cytoprotection of various normal tissues without attenuating antitumor response. The basis for the selectivity of action resides in the anabolism of amifostine at the normal tissue site by membrane-bound alkaline phosphatase. Dephosphorylation to the free thiol WR-1065 is followed by rapid uptake into normal tissues by a carrier-mediated facilitated diffusion process. In contrast, uptake into tumor tissues is slow to negligible. Pretreatment with amifostine provides protection of normal tissues from the cytotoxic effects of alkylating agents, organoplatinums, anthracyclines, taxanes, and radiation. Additionally, the mutagenic and carcinogenic effects of these modalities are also attenuated. Preclinical studies show significant protection of marrow progenitor cells that give rise to the red blood cells, white blood cells, and platelets. Protection of kidneys and neural tissues from cisplatin toxicity has been shown, along with protection of the heart, intestinal crypt cells, and pulmonary tissues from chemotherapy and radiation, as well as vasculoconnective and musculoconnective tissue in an irradiated field. Comparative in vitro and in vivo studies using murine and human tumor xenografts show no attenuation of antitumor effects of these same therapies despite the protection of normal organs. The unique preclinical profile of amifostine serves as the basis for the clinical development program for this important new broad-spectrum cytoprotective agent.

Pharmacokinetic profile of amifostine.

This article reviews the chemistry, measurement, metabolism, and pharmacokinetics of the cytoprotective agent amifostine. Validated analytic methodology to measure parent drug and pharmacologically active metabolites and pharmacokinetic studies are essential to the efficient performance and analysis of clinical studies. Well-validated analytic methods developed in the authors' laboratory were used to characterize this agent. Amifostine [s-2-(3-aminopropylamino)ethyl-phosphorothioate] is the phosphorylated pro-drug form of the active free thiol drug WR-1065 [2-(3-aminopropylamino)ethanethiol]. Observations described here support the hypothesis that amifostine is dephosphorylated rapidly by alkaline phosphatase present on the plasma membranes of the arteriolar endothelium of various normal tissues and on the plasma membranes of the kidneys' proximal tubular epithelium to its active thiol metabolite WR-1065, which in turn immediately enters normal tissues. Other metabolites that have been identified are WR-33278, the symmetrical disulfide of WR-1065; the mixed disulfides WR-1065-cysteine and WR-1065-glutathione; and cysteamine. Amifostine was recently approved by the US Food and Drug Administration for use as a cytoprotector in cancer patients receiving chemotherapy. Current pharmacokinetic studies in cancer patients are focusing on establishing a population model as a basis for developing limited sampling strategies for future investigations of the pharmacokinetic-pharmacodynamic behavior of amifostine.

Preclinical studies on the radioprotective efficacy and pharmacokinetics of subcutaneously administered amifostine.

The radioprotective effects and pharmacokinetics of subcutaneously (SC) administered amifostine have been investigated in animal studies. Studies in rats using a single dose of amifostine showed that SC administration gave protection from radiation-induced mucositis that is at least equivalent to that achieved by intravenous administration of the drug. These studies also indicate that tissue levels of the active metabolite WR-1065 correlated better with the radioprotective effects of amifostine than do plasma WR-1065 levels. Multiple-dose studies in rats show radioprotective effects equal to or greater than those obtained with intravenous dosing in the setting of fractionated irradiation. In addition, there is no evidence of drug accumulation in either normal or tumor tissue, with tumor WR-1065 levels peaking just above the limits of quantitation during treatment. Preliminary data from studies of SC amifostine in monkeys indicate a plasma pharmacokinetic profile similar to that reported earlier in humans. Tissue WR-1065 levels were higher at 30 minutes after SC dosing than they were after intravenous dosing and were comparable for the two routes at 60 minutes.

Down-regulation of intestinal-type alkaline phosphatase in the tumor vasculature and stroma provides a strong basis for explaining amifostine selectivity.

Strong clinical and experimental evidence indicates that amifostine confers cytoprotection in normal, but not in tumor, tissues. However, the mechanism of such selective action is poorly understood. Intestinal-type alkaline phosphatase (IAP) is a major isoenzyme involved in the hydrolysis of amifostine (WR-2721) to its active thiol form WR-I065. Could differences in IAP expression between normal and malignant tissues account for amifostine's selectivity? Paraffin-embedded material from normal breast, lung, colon, and head and neck tissues, together with their malignant counterparts, were retrieved and stained immunohistochemically for human IAP (antibody 7324, Abcam, Cambridge, UK) and endothelial cell CD31 antigen. Normal tissues (epithelium, fibroblasts, and vessels) consistently displayed strong nuclear and cytoplasmic IAP reactivity. The vascular density (number of positive vessels per x 200 optical field), whether assessed in anti-IAP or anti-CD31 stained sections, was very similar, indicating a strong IAP content for the entire normal vasculature. Therefore, amifostine hydrolysis is ensured in normal tissues and may occur at both vascular and interstitial levels. By contrast, 60% of the tumors analyzed showed a loss of IAP expression in both epithelial cells and stroma, and only 10% to 15% of them demonstrated nuclear/cytoplasmic reactivity, which was confined to the epithelial cells. Similarly, the percentage of tumor vessels exhibiting some IAP reactivity was very low (6% to 17%). This dramatic loss of IAP expression from tumor stroma/vasculature may form a strong basis for explaining amifostine selectivity. In contrast, the abundance of IAP expression in normal tissues, stromal and vascular, ensures an intense hydrolysis of WR-2721 and rapid intracellular accumulation of WR-1065.

A method for the combined measurement of ethiofos and WR-1065 in plasma: application to pharmacokinetic experiments with ethiofos and its metabolites.

An analytical method for the combined measurement of ethiofos (WR-2721) and a major metabolite (WR-1065) in plasma is described. Plasma samples were subjected to conditions which quantitatively converted both ethiofos and bound WR-1065 to free WR-1065 which was subsequently separated by HPLC and detected electrochemically using established procedures. Although bound WR-1065 in plasma is thought to exist mainly in the form of mixed disulfides, the symmetrical disulfide, WR-33278, also was quantitatively converted to the free thiol form. Standard curves were linear over the range 0.10 to 25 micrograms/mL (0.75 to 186 mumol/L). Mean precision over the range was 5.4% (coefficient of variation, CV) and recoveries of various mixtures of ethiofos, WR-1065 and WR-33278 averaged 102% (CV = 6.6%). This analytical procedure and others specific for ethiofos, free WR-1065 and WR-33278 were applied to dosing experiments in which the parent drug and its major metabolites were variously administered to beagle dogs and rhesus monkeys. Following i.v. administration of ethiofos (120-150 mg per kg body weight) to monkeys, plasma concentrations of unchanged drug ranged from 477 micrograms/mL (2.23 mM) down to the minimum detectable limit of the analytical procedure (0.05 micrograms/mL, 0.23 microM) 2-3 hours postinfusion. Clearances averaged 43.5 +/- 13.4 (SD) mL min-1 kg-1 and half-lives observed in the 20-60 minute postinfusion period were 8-15 min.

Human pharmacokinetics of WR-2721.

The pharmacokinetic properties of WR-2721 were investigated in 13 cancer patients given a 150 mg/M2 intravenous bolus dose of the drug. An average plasma clearance value of 2.17 L/min was obtained. Very little of the drug or the two metabolites, WR-1065 and WR-33278, were excreted in urine obtained after the blood collection schedule. Plasma concentrations of WR-2721 decreased by 94% within 6 minutes of drug administration. The mean value of 6.44 L obtained for the steady-state volume of distribution indicates that the extravascular space occupied by the drug is small. These observations suggest that in human cancer patients, WR-2721 is rapidly taken up by tissues and converted to metabolites.

Transplacental uptake of WR 2721 by the rat embryo.

On day 14 post-conception, near the end of the period of major organogenesis, pregnant rats were injected intravenously or intraperitoneally with WR 2721 spiked with 14C-WR 2721. The radioprotectant was shown to cross the placenta rapidly when administered by either route, and the concentration of WR 2721 in the embryos, placentae, and maternal blood plasma was determined during the period 5 to 90 minutes following administration. The concentration of WR 2721 increased continuously in the embryos during this period and did so against a decreasing concentration in the maternal blood. Injection of WR 2721 at 100 mg/kg of maternal body weight resulted in the presence of 8-9-mg/kg embryo weight; this embryo level is about 1/2 the injected dose of WR 2721 currently being used in human radiotherapy trials, that is, 20 mg/kg (740 mg/m2) body weight. Previous toxicity studies of 9, 11, and 14 day rat embryos have shown that this 100 mg/kg dose is much below the level which produces embryotoxic effects.

Pharmacokinetics of WR-1065 in mouse tissue following treatment with WR-2721.

Levels of reduced glutathione (GSH) and N-(2-mercaptoethyl)-1,3-diaminopropane (WR-1065) were measured in tissues of Balb/c mouse bearing EMT6 tumors at time intervals ranging from 5 min to 48 hr after i.v. injection of S-2-(3-aminopropylamino)ethyl phosphorothioate (WR-2721) at 500 mg per kg. In all tissues examined (liver, kidney, lung, heart, muscle, brain, tumor, spleen, and salivary gland), maximal WR-1065 levels occurred 5-15 min after injection, with levels in liver, kidney, lung, and salivary gland exceeding one mumole per gm. The post-maximum decline in WR-1065 varied markedly with tissue, lung exhibiting a 6-fold drop by 30 min and salivary gland falling only 15% after 3 hr. In a mouse treated with carbon-14 labeled WR-2721 it was found after 15 min that WR-1065 accounted for over half of the total drug in all tissues except tumor, where it accounted for a third of the total drug. There was no evidence that GSH levels were substantially altered by WR-2721 treatment. The results provide the first direct evidence supporting the widely held view that WR-2721 treatment results in intracellular WR-1065 and they demonstrate that high levels of WR-1065 occur very soon after i.v. injection.

Variation in normal tissue responsiveness to WR-2721.

The radioprotection afforded normal tissues by WR-2721 or S-2-[3-aminopropylamino]ethylphosphorothioic acid varies widely, with some of the most responsive tissues showing low levels of absorbed drug and vice versa. While the oxygen tension of the respective tissues may be contributing to this variation, it can be shown that the drug is not homogeneously distributed within each tissue, and that its location within the sub-cellular compartments can vary widely. Recognition of this variability, when combined with the newly developed HPLC assays for drug quality, should provide new insights into the sources of variation in normal tissue responsiveness to these agents.

Comparative biodistribution and radioprotection studies with three radioprotective drugs in mouse tumors.

The organ level biodistribution and tumor radioprotective properties of three drugs have been compared: WR-2721 (NSC 296961), WR-3689 (NSC 327729), and WR-77913 (NSC 318809). The three drugs have similar distribution patterns in normal mouse tissues. At 30 minutes after intraperitoneal injection, highest levels of 35S from radiolabeled protector are found in kidney and submandibular salivary gland, with lowest levels in brain and moderately low values in tumor and skin. Three of four tumors examined take up less WR-3689 than the other two protectors. For the three protectors, the dose modifying factors for the RIF-1 tumor irradiated in vivo and assayed in vitro are 1.5-1.7, but do not vary as predicted by differential uptake of drug into this neoplasm. In RIF-1, WR-3689 is taken up most avidly, but the three drugs tend to be equally protective.