The Impact of Dipyridamole on Disease-Associated Microglia Phenotypic Transformation in White Matter Lesions Induced by Chronic Cerebral Hypoperfusion.

White matter lesions (WMLs) resulting from chronic cerebral hypoperfusion (CCH) are the leading cause of vascular dementia (VaD). This study aimed to investigate whether dipyridamole could alleviate WMLs by regulating the phenotype of disease-associated microglia (DAM) through equilibrative nucleoside transporter 2 (ENT2) and adenosine A2A receptor (Adora2a) and to clarify the underlying molecular mechanisms. CCH rat models were constructed to mimic VaD. Morris water maze and Luxol Fast Blue staining were employed to assess cognitive function and quantify the severity of WMLs, respectively. Immunofluorescent staining was performed to analyze the activation of glial cells and the phenotypic transformation of DAM. Additionally, levels of ENT2, proteins in the NF-κB and ERK1/2 pathways and inflammatory cytokines were detected. The results indicated that dipyridamole diminished the activation and proliferation of microglia and astrocytes, increased the expression of myelin basic protein and ameliorated WMLs and cognitive decline in CCH rats. Further study revealed that dipyridamole decreased the expression of ENT2 and inhibited the activation of ERK1/2 and NF-κB signaling pathways, which ultimately converted DAM to anti-inflammatory phenotype and suppressed the levels of TNF-α, IL-1ß, IL-6 in WMLs. However, Adora2a inhibitor (SCH58261) attenuated above effects. Our study demonstrates that dipyridamole facilitates the conversion of DAM to the anti-inflammatory phenotype through ENT2/Adora2a pathway and inhibits the activation of ERK1/2 and NF-κB signaling pathways, thereby alleviating neuroinflammation in WMLs. The current findings establish the basis for using dipyridamole to treat VaD.

Construction and study of blood purification membrane modified with PDE inhibitor: Investigation of antiplatelet activity and hemocompatibility.

The recent "cell-based theory" of coagulation suggests that platelets serve as the site of coagulation factor reactions, making platelets an effective target for inhibiting membrane thrombosis. Unfortunately, there is limited research on how blood purification membranes affect platelet intracellular signaling. In this study, we modified polyethersulfone (PES) membranes with the platelet phosphodiesterase (PDE) inhibitor dipyridamole (DIP) and investigated the effects of the DIP/PES (DP) membranes on platelet adhesion, activation, aggregation, and secretion, as well as the role of the PDE-cyclic adenosine monophosphate (cAMP) intracellular signaling pathway. Additionally, we evaluated the hemocompatibility and preliminary in vivo safety of DP membranes. Our results demonstrate that the modified DP membranes effectively inhibited platelet adhesion, membrane CD62P expression, and plasma soluble P-selectin activation levels. Furthermore, we confirmed that DP membranes achieved platelet aggregation inhibition and reduced platelet factor 4 and ß-thromoglobulin secretion levels by inhibiting platelet intracellular PDE-cAMP signaling. Moreover, the modified DP membranes exhibited good anticoagulant and red blood cell membrane stability and complement resistance and demonstrated preliminary biocompatibility in mouse experiments. Collectively, these findings highlight the potential application of DP dialysis membranes in blood purification for critically ill patients.

Dipyridamole ameliorates doxorubicin-induced cardiotoxicity.

Dipyridamole is a platelet inhibitor with antithrombotic properties that can help prevent stroke recurrence. Twenty-eight male rats were divided randomly into four groups (7 rats in each group). Control group: rats received a natural diet and water. Normal saline group: rats received 0.9% normal saline for two weeks. Doxorubicin group (induced group): rats received 2.5 mg/kg three times a week for two weeks. Dipyridamole group (dipyridamole treated group): received dipyridamole (6 mg/kg/daily) orally for two weeks. Doxorubicin caused cardiotoxicity as indicated by a significant increase in tumor necrosis factor-α, interleukin-6, malondialdehyde, and caspase-3 level (P<0.05), while total antioxidant capacity and Bcl-2 levels were significantly reduced in cardiac tissues of rats in the doxorubicin group compared to the normal saline control group (P<0.05). Dipyridamole significantly ameliorates doxorubicin-induced cardiotoxicity, as suggested by a significant decrease in inflammatory markers (tumor necrosis factor-α and interleukin-6) (P<0.05). Moreover, the cardiac tissue level of oxidative marker malondialdehyde was significantly decreased (P<0.05), and total antioxidant capacity significantly increased in the dipyridamole group in comparison to the doxorubicin-only group (P<0.05). Dipyridamole exerted a significant heart-protective effect against doxorubicin-induced cardiotoxicity in rats, probably via interfering with oxidative stress, inflammatory response, and apoptotic pathway. The goal of this study was to investigate the potential protective effect of dipyridamole against doxorubicin-induced cardiotoxicity via interfering with pro-inflammatory, oxidative, and apoptotic pathways.

Effect of Dipyridamole on Membrane Energization and Energy Transfer in Chromatophores of Rba. sphaeroides.

Effect of dipyridamole (DIP) at concentrations up to 1 mM on fluorescent characteristics of light-harvesting complexes LH2 and LH1, as well as on conditions of photosynthetic electron transport chain in the bacterial chromatophores of Rba. sphaeroides was investigated. DIP was found to affect efficiency of energy transfer from the light-harvesting complex LH2 to the LH1-reaction center core complex and to produce the long-wavelength ("red") shift of the absorption band of light-harvesting bacteriochlorophyll molecules in the IR spectral region at 840-900 nm. This shift is associated with the membrane transition to the energized state. It was shown that DIP is able to reduce the photooxidized bacteriochlorophyll of the reaction center, which accelerated electron flow along the electron transport chain, thereby stimulating generation of the transmembrane potential on the chromatophore membrane. The results are important for clarifying possible mechanisms of DIP influence on the activity of membrane-bound functional proteins. In particular, they might be significant for interpreting numerous therapeutic effects of DIP.

Assessment of on-treatment platelet reactivity at high and low shear stress and platelet activation status after the addition of dipyridamole to aspirin in the early and late phases after TIA and ischaemic stroke.

BACKGROUND: Data are limited on the ability of dipyridamole to additionally inhibit platelet function/reactivity in ischaemic cerebrovascular disease (CVD) patients on aspirin. AIMS: To assess inhibition of platelet function/reactivity and platelet activation with dipyridamole in CVD. METHODS: This prospective, observational study assessed TIA/ischaemic stroke patients before (baseline; N = 60), at 14 ±7 days (14d, N = 39) and ≥ 90 days (90d, N = 31) after adding dipyridamole to aspirin. Platelet function/reactivity at high shear stress (PFA-100® C-ADP) and low shear stress (VerifyNow® P2Y12 and Multiplate® ADP assays), and platelet activation status (% expression of CD62P, CD63 and leucocyte-platelet complexes on whole blood flow cytometry) were quantified. 'Dipyridamole-high on-treatment platelet reactivity (HTPR)' was defined as failure to inhibit ADP-induced platelet aggregation +/- adhesion compared with the patient's baseline on aspirin monotherapy by more than twice the coefficient-of-variation of the assay after adding dipyridamole to aspirin. RESULTS: Dipyridamole-HTPR was identified in 71.4-75% of patients on PFA-100 C-ADP, 83.9-86.8% of patients on VerifyNow P2Y12, and 81.5-83.3% of patients on Multiplate ADP assays. There were no changes in CD62P/CD63 expression (P ≥ 0.18), or consistent changes in leucocyte-platelet complexes in CVD patients overall at 14d or 90d vs. baseline after commencing dipyridamole. Monocyte-platelet complexes increased in the patient subgroup with dipyridamole-HTPR at 14d and 90d on PFA-100, and at 14d on VerifyNow (P ≤ 0.04), but not in those without dipyridamole-HTPR. DISCUSSION: Additional antiplatelet effects of dipyridamole are detectable under high and low shear stress conditions with user-friendly platelet function/reactivity tests ex vivo. Increasing circulating monocyte-platelet complexes over time are associated with dipyridamole-HTPR.

Ginkgo leaf extract and dipyridamole injection for chronic cor pulmonale: a PRISMA-compliant meta-analysis of randomized controlled trials.

Ginkgo leaf extract and dipyridamole injection (GLED), a kind of Chinese herbal medicine preparation, has been considered as a promising supplementary treatment for chronic cor pulmonale (CCP). Although an analysis of the published literature has been performed, the exact effects and safety of GLED have yet to be systematically investigated. Therefore, a wide-ranging systematic search of electronic databases from which to draw conclusions was conducted. All randomized controlled trials concerning the GLED plus conventional treatments for CCP were selected in the present study. Main outcomes were treatment efficacy, blood gas and hemorrheology indexes, and adverse events. Data from 28 trials with 2457 CCP patients were analyzed. The results indicated that, compared with conventional treatments alone, the combination of conventional treatments with GLED obviously improved the markedly effective rate (RR = 1.44, 95% CI = 1.31-1.58, P < 0.00001) and total effective rate (RR = 1.28, 95% CI = 1.18-1.38, P < 0.00001). Moreover, the hemorrheology (PaO2, P < 0.00001; PaCO2, P < 0.00001; SaO2, P < 0.00001; pH value, P = 0.05) and blood gas indexes (PV, WBHSV, WBMSV, WBLSV, hematocrit and FBG, P < 0.01) of CCP patients were also significantly ameliorated after the combined therapy. The frequency of adverse events did not differ significantly between the two groups (P > 0.05). In summary, evidence from the meta-analysis suggested that the combination of conventional treatments and GLED appeared to be effective and relatively safe for CCP. Therefore, GLED mediated therapy could be recommended as an adjuvant treatment for CCP.

Involvement of nucleoside transporters in the transcorneal permeation of topically instilled substrates in rabbits in-vivo.

The objective of the current study was to characterize and evaluate the functional importance of the Nucleoside Transporters (NTs) in the cornea of the rabbits. Reverse transcriptase polymerase chain reaction (RT-PCR) was used for the molecular characterization of the NTs. Their functionality was evaluated using two substrates, ribavirin and cytarabine. Dipyridamole was used as a blocker for the study. All the treatments were given topically. Molecular characterization of NTs revealed presence of ent1, ent2, ent3 and cnt3 in the cornea. The concentration vs time profile for cytarabine in Aqueous Humor (AH) exhibited a statistically significant (p<0.05) drop at 1h with blocker pretreatment. The mean AUC0-2 between the treatments was also differing in a significant (p<0.05) manner. The concentration vs time profile for ribavirin in AH also showed a significant (p<0.05) decrease in its concentration at 1h with blocker pretreatment. Dipyridamole was able to block ribavirin's entry with as low as 40nM concentration while complete blockade was achieved at 8mM and above. When cytarabine and ribavirin were co-administered, ribavirin at a concentration of 6.5mM significantly inhibited (p<0.05) the transcorneal permeation of cytarabine up to 80%. To conclude, this study showed the presence and functional importance of NTs in the transcorneal uptake of nucleoside substrates. This study further revealed the presence of concentration dependent competitive inhibition among substrates for their transcorneal permeation.

Influence of nitric oxide and phosphodiesterases during in vitro maturation of bovine oocytes on meiotic resumption and embryo production.

This study aimed to examine the effects of nitric oxide (NO) and different phosphodiesterase (PDE) families on meiosis resumption, nucleotides levels and embryo production. Experiment I, COCs were matured in vitro with the NO donor S-nitroso-N-acetylpenicillamine (SNAP) associated or not with the soluble guanylate cyclase (sGC) inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ), meiotic resumption and nucleotides levels were assessed. SNAP delayed germinal vesicle breakdown (GVBD) (53.4 ± 1.2 versus 78.4 ± 2.4% for controls, P 0.05). Cyclic GMP levels were higher in SNAP (3.94 ± 0.18, P 0.05). Embryo development did not differ from the control for SNAP and cilostamide groups (38.7 ± 5.8, 37.9 ± 6.2 and 40.5 ± 5.8%, P > 0.05), but SNAP + cilostamide decreased embryo production (25.7 ± 6.9%, P < 0.05). In conclusion, SNAP was confirmed to delay meiosis resumption by the NO/sGC/cGMP pathway, by increasing cGMP, but not cAMP. Inhibiting different PDEs to further increase nucleotides in association with SNAP did not show any additive effects on meiosis resumption, indicating that other pathways are involved. Moreover, SNAP + cilostamide affected the meiosis progression and decreased embryo development.

Generation of radical form of dipyridamole at illumination of photosynthetic reaction centers of Rb. sphaeroides.

The study of the effect of vasodilator, antiplatelet agent, and inhibitor P-glycoprotein dipyridamole (DIP) on the functioning of the transmembrane protein of the reaction center (RC) of Rb. sphaeroides showed that the activation of RC by constant light generates the DIP radical cation, which significantly affects the kinetics of recombination of charges divided between photoactive bacteriochlorophyll and quinone acceptors. Thus, the antioxidant properties of DIP may affect the functional activity of membrane proteins, and this apparently should be taken into account in the studies of the mechanisms of therapeutic action of this drug.

Targeting the epigenetics of the DNA damage response in breast cancer.

Cancer is as much an epigenetic disease as it is a genetic disease, and epigenetic alterations in cancer often serve as potent surrogates for genetic mutations. Because the epigenetic factors involved in the DNA damage response are regulated by multiple elements, therapies to target specific components of the epigenetic machinery can be inefficient. In contrast, therapies aimed at inhibiting the methionine cycle can indirectly inhibit both DNA and protein methylation, and the wide variety of genes and pathways that are affected by these methylations make this global strategy very attractive. In the present study, we propose an adjuvant therapy that targets the epigenetics of the DNA damage response in breast cancer cells and that results in efficient apoptosis and a reduction in distant metastases in vivo. We observed that a combined therapy designed to uncouple adenosine metabolism using dipyridamole in the presence of a new synthetic antifolate, 3-O-(3,4,5-trimethoxybenzoyl)-(-)-catechin, simultaneously and efficiently blocked both the folic cycle and the methionine cycle in breast cancer cells and sensitized these cells to radiotherapy. The treatment impeded the recruitment of 53BP1 and BRCA1 to the chromatin regions flanking DNA double-strand breaks and thereby avoided the DNA damage responses in breast cancer cells that were exposed to ionizing radiation. In addition, this hypomethylating therapy was also efficient in reducing the self-renewal capability of breast cancer-initiating cells and induced reversion of mesenchymal phenotypes in breast cancer cells.

Prediction of the precipitation profiles of weak base drugs in the small intestine using a simplified transfer ("dumping") model coupled with in silico modeling and simulation approach.

BACKGROUND: Precipitation of poorly soluble, weakly basic drugs upon entering the small intestine may lead to poor bioavailability. It would be useful to be able to predict the extent of in vivo precipitation so that formulation measures to counteract this problem can be taken. AIM: The aim of this research was to characterize the precipitation kinetics of two representative weak base drugs, dipyridamole and ketoconazole in vitro using a simplified transfer model approach, and to establish a predictive model for the total and dissolved concentrations in the small intestine after oral administration using in silico modeling and simulation. METHODS: A simplified transfer ("dumping") method based on the USP paddle apparatus was used to obtain the precipitation profiles of the two weak base drugs by adding a solution of the drug in 0.02N hydrochloric acid to FaSSIF-V2. The observed precipitation curves obtained with various initial concentrations were fitted to first order kinetics. An in silico pharmacokinetic model for weak base drugs with precipitation in the small intestine was designed using STELLA® software and coupled with the precipitation profiles in order to simulate the total and dissolved drug concentrations in the small intestinal lumen in the fasted state in humans. RESULTS: The predicted total and dissolved concentration curves in small intestine for the two weak base drugs were similar to the concentration profiles observed in vivo. The fraction precipitated of the drugs in the small intestine was also well predicted, although the precipitation of ketoconazole at higher initial concentrations was somewhat overestimated. A sensitivity analysis conducted on the simulation of the precipitation of the drugs indicated that a higher fraction precipitated when gastric emptying was faster and/or the concentration of the drug in the added solution was higher. CONCLUSION: The dumping method provides a useful screen for precipitation in the small intestine, especially in the context of early development.

Effects of gastric pH on oral drug absorption: In vitro assessment using a dissolution/permeation system reflecting the gastric dissolution process.

The aim of the present study was to evaluate the effects of gastric pH on the oral absorption of poorly water-soluble drugs using an in vitro system. A dissolution/permeation system (D/P system) equipped with a Caco-2 cell monolayer was used as the in vitro system to evaluate oral drug absorption, while a small vessel filled with simulated gastric fluid (SGF) was used to reflect the gastric dissolution phase. After applying drugs in their solid forms to SGF, SGF solution containing a 1/100 clinical dose of each drug was mixed with the apical solution of the D/P system, which was changed to fasted state-simulated intestinal fluid. Dissolved and permeated amounts on applied amount of drugs were then monitored for 2h. Similar experiments were performed using the same drugs, but without the gastric phase. Oral absorption with or without the gastric phase was predicted in humans based on the amount of the drug that permeated in the D/P system, assuming that the system without the gastric phase reflected human absorption with an elevated gastric pH. The dissolved amounts of basic drugs with poor water solubility, namely albendazole, dipyridamole, and ketoconazole, in the apical solution and their permeation across a Caco-2 cell monolayer were significantly enhanced when the gastric dissolution process was reflected due to the physicochemical properties of basic drugs. These amounts resulted in the prediction of higher oral absorption with normal gastric pH than with high gastric pH. On the other hand, when diclofenac sodium, the salt form of an acidic drug, was applied to the D/P system with the gastric phase, its dissolved and permeated amounts were significantly lower than those without the gastric phase. However, the oral absorption of diclofenac was predicted to be complete (96-98%) irrespective of gastric pH because the permeated amounts of diclofenac under both conditions were sufficiently high to achieve complete absorption. These estimations of the effects of gastric pH on the oral absorption of poorly water-soluble drugs were consistent with observations in humans. In conclusion, the D/P system with the gastric phase may be a useful tool for better predicting the oral absorption of poorly water-soluble basic drugs. In addition, the effects of gastric pH on the oral absorption of poorly water-soluble drugs may be evaluated by the D/P system with and without the gastric phase.

Instrumentation of Flow-Through USP IV Dissolution Apparatus to Assess Poorly Soluble Basic Drug Products: a Technical Note.

Supersaturation and precipitation are common limitations encountered especially with poorly soluble basic drugs. The aims of this work were to explore the pattern of dissolution and precipitation of poorly soluble basic drugs using a United States Pharmacopoeia (USP) IV dissolution apparatus and to compare it to the widely used USP II dissolution apparatus. In order to investigate the influence of gastric emptying time on bioavailability, tables of two model drugs (dipyridamole 100 mg and cinnarizine 15 mg) were investigated and pH change from 1.2 to 6.8 were achieved after 10, 20 or 30 min using USP II or USP IV dissolution apparatuses. Using USP II, dipyridamole and cinnarizine concentrations dropped instantly as a result of drug precipitation with drug crystals evident in the dissolution vessel. At pH change times of 10, 20 and 30 min, the total amount of dissolved drug was dependent on pH change time. Using USP IV, at a flow rate of 8 ml/min, it was possible to have comparable release to agitation at 50 rpm using USP II suggesting that comparable hydrodynamic forces are possible. No drop in drug percentage occurs as the dissolved fraction was readily emptied from the flow cell, preventing drug accumulation in the dissolution medium. However, a negligible percentage of drug release took place following pH change. In conclusion, the use of the flow-through cell dissolution provided laminar flow, use of realistic fluid volumes and avoided precipitation of dissolved drug fraction in the gastric phase as it is discharged before pH change.

An in vitro biorelevant gastrointestinal transfer (BioGIT) system for forecasting concentrations in the fasted upper small intestine: Design, implementation, and evaluation.

PURPOSE: Design an in vitro methodology for studying gastrointestinal transfer in the fasted state and implement the methodology in vitro by using a biorelevant gastrointestinal transfer system(BioGIT); evaluate the usefulness of BioGIT in predicting luminal concentrations of lipophilic weak bases in the fasted upper small intestine. METHODS: The methodology was designed after modeling existing luminal data. Its implementation in vitro was based on a three compartment setup. Reproducibility of the transfer process was evaluated under conditions where solutions and/or suspensions were present in gastric and/or duodenal compartment and by using ranitidine, dipyridamole, ketoconazole, and posaconazole as model drugs. The transfer process as well as concentrations of dipyridamole, ketoconazole and posaconazole measured in the duodenal compartment were compared with data previously collected in the upper small intestine, after administration of identical preparations/dosage forms to fasted adults. RESULTS: Using BioGIT, the transfer process was performed reproducibly in all cases (RSD b 12.9%); data with dipyridamole and ketoconazole were in line with luminal data in humans. Dipyridamole, ketoconazole and posaconazole concentrations in duodenal compartment were also in line with previously measured concentrations in the fasted upper small intestine of healthy adults. CONCLUSIONS: BioGIT system could be useful for the evaluation of the impact of gastrointestinal transfer on concentrations in the upper intestinal lumen during the first hour, after oral administration of dispersing/solution dosage forms of lipophilic weak bases.

In vivo predictive mini-scale dissolution for weak bases: Advantages of pH-shift in combination with an absorptive compartment.

The purpose was the evaluation of a new miniscale biphasic dissolution model with pH-shift (miBIdi-pH). Its capability to predict supersaturation and precipitation of weak bases (e.g. dipyridamole) and the in vivo performance of various formulations of the model compound BIXX (weak base, poor solubility, good permeability) was investigated with respect to dissolution, precipitation and re-dissolution. Single phase dissolution with and without pH-shift [small scale dissolution (V = 20 ml) and USPII] and miBIdi-pH (50 ml aqueous phase covered by 15 ml octanol) were used for analyzing crystalline dipyridamole and the four BIXX-containing formulations. Precipitate was analyzed via X-ray diffraction. Bioavailability of the formulations was tested in dogs. Phoenix WinNonlin(®) was used for IVIVC. For dipyridamole, precipitation upon pH shift was less pronounced in the miBIdi-pH in comparison to the single phase dissolution (35% vs. 90%). In case of four BIXX-containing formulations, USPII revealed significant differences in their dissolution, whereas the final amounts of BIXX in the octanol phase in the miBIdi-pH were alike. Different partitioning rates into octanol were observed. The miBIdi-pH was superior to single phasic dissolution in predicting in vivo precipitation of dipyridamole. In case of the BIXX-containing formulations, it was superior in ranking the formulations and it was capable to capture the kinetics of different absorption processes in vivo.

Genetic background determines inflammatory angiogenesis response to dipyridamole in mice.

Inflammation and angiogenesis, key components of fibrovascular tissue growth, exhibit considerable variability among species and strains. We investigated whether the response of inbred and outbred mice strains to dipyridamole (DP) on these processes would present similar variability. The effects of the drug on blood vessel formation, inflammatory cell recruitment, collagen deposition and cytokine production were determined on the fibroproliferative tissue induced by sponge implants in Swiss and Balb/c mice. Angiogenesis as assessed by hemoglobin (Hb) and vascular endothelial growth factor (VEGF) concentrations differed between the strains. Swiss implants had the highest Hb content but the lowest VEGF concentrations. Systemic DP treatment exerted an antiangiogenic effect on Balb/c implants but an proangiogenic effect on Swiss implants. The inflammatory enzyme activities myeloperoxidase (six-fold higher in Balb/c implants) and N-acetyl-ß-D-glucosaminidase were reduced by the treatment in Balb/c implants only. Nitrite concentrations were also higher in Balb/c implants by 40% after DP treatment. Tumor necrosis factor-alpha levels were similar in the implants of both strains and were not reduced by DP. Transforming growth factor ß-1 levels and collagen deposition also varied between the strains. The inbred strain had similar levels of the cytokine but implants of Swiss mice presented more collagen. DP treatment reduced collagen deposition in Balb/c implants only. Our data showing the influence of the genetic background on marked heterogeneity of inflammatory angiogenesis components and differential sensitivity to DP may provide some answers to clinical evidence for resistance to angiogenic therapy.

An in vitro methodology for forecasting luminal concentrations and precipitation of highly permeable lipophilic weak bases in the fasted upper small intestine.

PURPOSE: To develop an in vitro methodology for prediction of concentrations and potential precipitation of highly permeable, lipophilic weak bases in fasted upper small intestine based on ketoconazole and dipyridamole luminal data. Evaluate usefulness of methodology in predicting luminal precipitation of AZD0865 and SB705498 based on plasma data. METHODS: A three-compartment in vitro setup was used. Depending on the dosage form administered in in vivo studies, a solution or a suspension was placed in the gastric compartment. A medium simulating the luminal environment (FaSSIF-V2plus) was initially placed in the duodenal compartment. Concentrated FaSSIF-V2plus was placed in the reservoir compartment. RESULTS: In vitro ketoconazole and dipyridamole concentrations and precipitated fractions adequately reflected luminal data. Unlike luminal precipitates, in vitro ketoconazole precipitates were crystalline. In vitro AZD0865 data confirmed previously published human pharmacokinetic data suggesting that absorption rates are not affected by luminal precipitation. In vitro SB705498 data predicted that significant luminal precipitation occurs after a 100 mg or 400 mg but not after a 10 mg dose, consistent with human pharmacokinetic data. CONCLUSIONS: An in vitro methodology for predicting concentrations and potential precipitation in fasted upper small intestine, after administration of highly permeable, lipophilic weak bases in fasted upper small intestine was developed and evaluated for its predictability in regard to luminal precipitation.

Metabolism and accumulation of the lipophilic deoxynucleoside analogs elacytarabine and CP-4126.

Cytarabine (ara-C) and gemcitabine (dFdC) are commonly used anticancer drugs, which depend on the equilibrative (ENT) and concentrative-nucleoside-transporters to enter the cell. To bypass transport-related drug resistance, lipophilic derivatives elacytarabine (CP-4055), ara-C-5'elaidic-acid-ester, and CP-4126, (CO 1.01) gemcitabine-5'elaidic-acid-ester, were investigated for the entry into the cell, distribution, metabolism and retention. The leukemic CEM-cell-line and its deoxycytidine-kinase deficient variant (CEM/dCK-) were exposed for 30 and 60 min to the radiolabeled drugs; followed by culture in drug-free medium in order to determine drug retention in the cell. The cellular fractions were analyzed with thin-layer-chromatography and HPLC. Elacytarabine and CP-4126 were converted to the parent compounds both inside and outside the cell (35-45%). The ENT-inhibitor dipyridamole did not affect their uptake or retention. Inside the cell Elacytarabine and CP-4126 predominantly localized in the membrane and cytosolic fraction, leading to a long retention after removal of the medium. In contrast, in cells exposed to the parent drugs ara-C and dFdC, intracellular drug concentration increased during exposure but decreased to undetectable levels after drug removal. In the dCK- cell line, no metabolism was observed. The concentrations of ara-CTP and dFdCTP reached a peak at the end of the incubation with the drugs, and decreased after drug removal; peak levels of dFdCTP were 35 times higher than ara-CTP and was retained better. In contrast, after exposure to elacytarabine or CP-4126, ara-CTP and dFdCTP levels continued to increase not only during exposure but also during 120 min after removal of the elacytarabine and CP-4126. Levels of ara-CTP and dFdCTP were higher than after exposure to the parent drugs. In conclusion, the lipophilic derivatives elacytarabine and CP-4126 showed a nucleoside-transporter independent uptake, with long retention of the active nucleotides. These lipophilic nucleoside analogues are new chemical entities suitable for novel clinical applications.

Isolation and characterisation of degradant impurities in dipyridamole formulation.

Dipyridamole is an antithrombotic drug. In the stability study of drug product of Dipyridamole, two unknown impurities (referred as DP-I and DP-II) were detected at levels of 0.25% and 0.54% by gradient reverse phase HPLC method. The drug product was subjected to stress to enhance the level of these impurities. An elegant isocratic preparative method was employed using a Reprosil CN column with a short run time of 14 min to isolate these impurities. The DP-I and DP-II were isolated with purities of 99.1% and 99.8% respectively. Structural studies of these impurities were undertaken using spectroscopic techniques such as IR, NMR and Mass. Based on the spectral data, the structures of DP-I and DP-II have been characterised to be 2,2',2″,2'″-(4-hydroxy-8-(piperidin-1-yl) pyrimido [5,4-d]pyrimidine-2,6 diyl) bis(azanetriyl) tetraethanol, 4-(2-((6-(bis (2-hydroxyethyl) amino)-4, 8-di (piperidin-1-yl) pyrimido [5,4-d] pyrimidin-2-yl) (2-hydroxyethyl) amino) ethoxy)-2, 3-dihydroxy-4-oxobutanoic acid, respectively. A detailed elucidation of the structure is presented in this article.

Effects of acute post-treatment with dipyridamole in a rat model of focal cerebral ischemia.

Dipyridamole (DP) is a platelet inhibitor with known antithrombotic benefits in stroke prevention. In addition to its anti-aggregant properties, recent studies have reported that DP promotes anti-inflammatory, anti-oxidative and neuroprotective effects. We aimed to test whether post-treatment with DP may exert protection after ischemic cerebral injury in the rat. For this purpose, rats were subjected to 120 min or 90 min of middle cerebral artery occlusion (MCAO) followed by 24 or 48 h of reperfusion, respectively. Either DP (100mg/kg) or vehicle was administered i.v. at the onset of reperfusion; rats subjected to 90 min MCAO also received additional doses of DP orally (60 mg/kg) at 24 and 36 h after ischemia. Matrix metalloproteinases, extravasated hemoglobin content and IL-6, MIP-1α and MCP-1 cytokine level were examined in brain tissue by zymography, western blot and multiple ELISA, respectively. DP post-treatment led to a neurological improvement in both models (p < 0.05) and a significant reduction in the infarct volume of rats subjected to 90 min of ischemia, as compared to vehicle group (7.9% vs. 24.4%, p = 0.03). This neuroprotection was accompanied by a modest increase in expression of MMP-9 pro-form and a significant attenuation of MIP-1α levels in the infarcted hemisphere. These results provide support for the development of novel therapies based on DP for acute treatment of stroke. In selected animals, intravenous administration of high dose DP induced an adverse hypotensive effect leading to rapid death. Thus, alternative ways of acute administration must be examined in order to avoid this unfavorable effect.