Post-Exercise Hypotension: An Alternative Management Strategy for Hypertension and Cardiovascular Disease?

Cardiovascular disease (CVD), including hypertension, is a leading cause of death worldwide and imposes an enormous burden on our societies [...].

Hemodynamic Assessment and In vivo Catabolism of Adenosine 5'-triphosphate in Doxorubicin or Isoproterenol-induced Cardiovascular Toxicity.

OBJECTIVE: Previous studies have shown that catabolism of adenosine 5'-triphosphate (ATP) in systemic blood is a potential surrogate biomarker for cardiovascular toxicity. We compared the acute toxicity of high doses of doxorubicin (DOX) and isoproterenol (ISO) on hemodynamics and ATP catabolism in the systemic circulation. METHODS: sprague Dawley (SD) rats (n = 8 - 11) were each given either a single dose of 30 mg/kg ISO, or a twice-daily dose of 10 mg/kg of DOX or 4 doses of normal saline (control) by subcutaneous injection. Blood samples were collected up to 6 hours for measuring concentrations of ATP and its catabolites. Hemodynamics was recorded continuously. The difference was considered significant at p < 0.05 (ANOVA). RESULTS: Mortality was 1/8, 5/11, and 0/11 for the DOX, ISO, and control groups, respectively. Systolic blood pressure was significantly lower in the DOX and ISO treated rats than in control measured at the last recorded time (76 ± 9 for DOX vs. 42 ± 8 for ISO vs. 103 ± 5 mmHg for control, p < 0.05 for all). Blood pressure fell gradually after the final injection for both DOX and control groups, but abruptly after ISO, followed by a rebound and then gradual decline till the end of the experiment. Heart rate was significantly higher after ISO, but there were no differences between the DOX and control rats (p > 0.05). RBC concentrations of ADP and AMP, and plasma concentrations of adenosine and uric acid were significantly higher in the ISO group. In contrast, hypoxanthine concentrations were significantly higher in the DOX treated group (p < 0.05). CONCLUSION: Acute cardiovascular toxicity induced by DOX and ISO may be measured by changes in hemodynamics and breakdown of ATP and adenosine in the systemic circulation, albeit a notable qualitative and quantitative difference was observed.

Adenosine 5'-Triphosphate Metabolism in Red Blood Cells as a Potential Biomarker for Post-Exercise Hypotension and a Drug Target for Cardiovascular Protection.

The importance of adenosine and ATP in regulating many biological functions has long been recognized, especially for their effects on the cardiovascular system, which may be used for management of hypertension and cardiometabolic diseases. In response to ischemia and cardiovascular injury, ATP is broken down to release adenosine. The effect of adenosine is very short lived because it is rapidly taken up by erythrocytes (RBCs), myocardial and endothelial cells, and also rapidly catabolized to oxypurine metabolites. Intracellular adenosine is phosphorylated back to adenine nucleotides via a salvage pathway. Extracellular and intracellular ATP is broken down rapidly to ADP and AMP, and finally to adenosine by 5′-nucleotidase. These metabolic events are known to occur in the myocardium, endothelium as well as in RBCs. Exercise has been shown to increase metabolism of ATP in RBCs, which may be an important mechanism for post-exercise hypotension and cardiovascular protection. The post-exercise effect was greater in hypertensive than in normotensive rats. The review summarizes current evidence in support of ATP metabolism in the RBC as a potential surrogate biomarker for cardiovascular protection and toxicities. It also discusses the opportunities, challenges, and obstacles of exploiting ATP metabolism in RBCs as a target for drug development and precision medicine.

Adenosine and Adenosine 5'-triphosphate Catabolism in Systemic Blood as a Potential Biomarker for Doxorubicin Cardiotoxicity in an Experimental Rat Model in vivo.

BACKGROUND: Previous studies have shown that metabolism of adenosine 5'-triphosphate (ATP) in systemic blood is a potential surrogate biomarker for cardiovascular toxicity. OBJECTIVE: To investigate the acute effect of high dose of doxorubicin (DOX) on adenosine and ATP catabolism in systemic blood in vivo. METHOD: Sprague Dawley (SD) rats were each given either 10 mg/kg of DOX (n = 8) or normal saline (1 mL/kg, n = 11) twice daily for 4 doses by subcutaneous (sc) injection. Blood samples were collected sequentially for up to 6 hours for measuring circulating concentrations of ATP, adenosine and their metabolites. Hemodynmic recording was obtained continuously after the last injection. The difference in response between groups was considered significant at p < 0.05 (t-test). RESULTS: Diastolic blood pressure (DBP) was significantly lower in the DOX treated rats than in the control before the final injection (87 ± 12 vs. 104 ± 11 mmHg, p < 0.05). Blood pressure fell gradually after the last injection and the decrease was significantly greater in the DOX treated group (p < 0.05). Plasma concentration of adenosine was significantly lower in the DOX treated group. In contrast, plasma concentrations of uric acid and hypoxanthine, as well as Red Blood Cell (RBC) concentrations of AMP, were significantly higher (p < 0.05). CONCLUSION: Acute cardiotoxicity induced by DOX may be measured by the increased breakdown of ATP to AMP in the RBC and also breakdown of adenosine to hypoxanthine and uric acid in plasma.

Metabolomics and Biomarkers for Drug Discovery.

Metabolomics and biomarkers are increasingly used in drug discovery and development, and are applied to personalized medicine. Progress in these research areas has increased our understanding of disease pathology and improved therapeutic strategies for many diseases with unmet challenges. Further advances will ultimately result in the development of better drugs and breakthrough therapies, which will benefit millions of patients suffering from chronic and life-threatening diseases worldwide.

Effect of Cardiovascular Injury on Catabolism of Adenosine and Adenosine 5-'Triphosphate in Systemic Blood in a Freely Moving Rat Model In Vivo.

BACKGROUND: Previous studies have shown that catabolism of adenosine 5'-triphosphate (ATP) in red blood cell (RBC) may be a key factor for cardiovascular protection and maintaining cardiovascular homeostasis. OBJECTIVE: To investigate the effect of cardiovascular injury on adenosine and ATP catabolism in systemic blood using a freely moving rat model in vivo. METHOD: After acclimatized to the experimental environment, Sprague Dawley (SD) rats were each given either isoproterenol (30 mg/kg) or saline (1 mL/kg) by subcutaneous (sc) injection. Blood samples were collected sequentially for up to 6 hours for measurement of red blood cell (RBC) concentrations of adenine nucleotides and plasma concentrations of adenosine and its oxypurine metabolites. RESULTS: We have found isoproterenol induced 50% mortality under the experimental condition. Plasma concentrations of adenosine (ADO) and uric acid (UA) and red blood cell (RBC) concentrations of adenosine 5'-diphosphate (ADP) and adenosine 5'-monophosphate (AMP) in RBC were significantly higher in the isoproterenol treated rats (p < 0.05 for all the comparison). On the other hand, plasma concentrations of hypoxanthine (HYP) were higher in the control group (p < 0.05), but there was no statistically significant changes in ATP concentrations in the RBC (p > 0.05). CONCLUSION: Cardiovascular injury induced by isoproterenol resulted in breakdown of ATP to ADP and AMP in the RBC and also breakdown of ADO to UA in plasma and other tissues.

A Pilot Study to Assess Adenosine 5'-triphosphate Metabolism in Red Blood Cells as a Drug Target for Potential Cardiovascular Protection.

OBJECTIVE: To study the effect of exercise preconditioning on adenosine 5'triphosphate (ATP) metabolism in red blood cells and cardiovascular protection against injury induced by isoproterenol in vivo. METHODS: Male Sprague Dawley rats (SDR) were each exercised on a treadmill for 15 minutes at 10 m/min and 10% grade (n = 7) (LowEx), or 14 m/min and 22% grade (n = 8) (VigEx). Two hours after the exercise, each rat received a single dose of isoproterenol (30 mg/kg) by subcutaneous (sc) injection. Two separate groups of SDR were used as control: One received no exercise (n = 10) (NoEx) and the other received no exercise and no isoproterenol (n = 11) (NoIso). Serial blood samples were collected over 5 hours for measurement of ATP and its catabolites by a validated HPLC. Hemodynamic recording was collected continuously for the duration of the experiment. Data were analysed using ANOVA and t-tests and difference considered significant at p < 0.05. RESULTS: Exercise pre-conditioning (both LowEx and VigEx) reduced mortality after isoproterenol from 50% to < 30% (p > 0.05). It attenuated the rebound in blood pressure significantly (p < 0.05 between NoEx vs VigEx), attenuated the increase of RBC adenosine 5'-monophosphate (AMP) concentrations induced by isoproterenol, and also decreased the breakdown of ATP to AMP in the RBC (p < 0.05 vs NoEx). CONCLUSION: Exercise pre-conditioning decreased the blood pressure rebound and also breakdown of ATP in RBC after isoproterenol which may be exploited further as a drug target for cardiovascular protection and prevention.

Diltiazem reduces mortality and breakdown of ATP in red blood cell induced by isoproterenol in a freely moving rat model in vivo.

The benefit of calcium channel blockers for cardiovascular prevention against heart attack and stroke has not been firmly supported. We investigated the possible cardiovascular protective effect of diltiazem (DTZ) against injury induced by isoproterenol using a freely moving rat model in vivo. Sprague Dawley rats were injected subcutaneously (sc) with either 5 or 10 mg/kg of DTZ, or saline as control, twice daily for five doses. One hour after the last injection, a single dose of isoproterenol (30 mg/kg) was injected sc to each rat. Blood samples were collected serially for 6 h for measurement of adenine nucleotides (ATP, ADP and AMP) in red blood cell (RBC) by a validated HPLC. The study has shown isoproterenol induced 50% mortality and also increased RBC concentrations of AMP from 0.04 ± 0.02 to 0.29 ± 0.21 mM at the end of the experiment (p < 0.05). Treatment with 10 mg/kg of DTZ reduced mortality from 50% to <20% and attenuated the increase of RBC concentrations of AMP from +0.25 ± 0.22 in the control rats to +0.072 ± 0.092 mM (p < 0.05). The study concluded that 10 mg/kg of DTZ reduced mortality and breakdown of ATP induced by isoproterenol in rats.

Effect of acute exercise on cardiovascular hemodynamic and red blood cell concentrations of purine nucleotides in hypertensive compared with normotensives rats.

OBJECTIVE: The mechanisms of exercise-induced health benefits are complex and not fully understood. This study investigated the effects of exercise and hypertension on cardiovascular hemodynamic responses and red blood cell (RBC) concentrations of purine nucleotides using normotensive and hypertensive rat models in vivo. METHODS: Sprague Dawley rats (SDRs) and spontaneously hypertensive rats (SHRs) were exercised on a treadmill for 15 min at a speed of 10 m/min and 5% grade. Blood samples were obtained from each rat before, during, and after exercise for measurement of adenosine 5'-triphosphate (ATP) and guanosine 5'-triphosphate (GTP) concentrations in RBCs by a validated high-performance liquid chromatography assay. They were returned to a restrainer after exercise, and hemodynamic recording collected continuously up to 6 h. Two separate groups (SDRs and SHRs) without exercise were used as controls. Biomarker data were compared between SDRs and SHRs using analysis of variance and t test and difference considered significant at p < 0.05. RESULTS: The study has demonstrated for the first time a difference in the postexercise effect between SDRs and SHRs. The 15 min of exercise significantly increased systolic blood pressure (SBP) (129 ± 16 to 162 ± 26 mmHg) and heart rate (HR) (416 ± 29 to 491 ± 26 bpm) in SDRs (p < 0.05), but not in SHRs. The postexercise hemodynamic effects were more profound in SHRs. SBP and diastolic blood pressure (DBP) also fell significantly in the control group of SHRs (SBP 184 ± 14 to 152 ± 29 mmHg and DBP 149 ± 9 to 120 ± 14 mmHg, p < 0.05 for both) towards the end of the experiment but not in the SDR group. The RBC concentrations of ATP and GTP increased after exercise in both SDRs and SHRs which were significantly correlated with the postexercise hemodynamic effect (p < 0.05). CONCLUSION: SHRs were more tolerant to increases in HR and SBP induced by exercise, and have more profound postexercise hemodynamic effects than SDRs. The hemodynamic effects were linked closely with RBC concentrations of ATP and GTP in both SDRs and SHRs.

Comparing pharmacokinetics and metabolism of diltiazem in normotensive Sprague Dawley and Wistar Kyoto rats vs. spontaneously hypertensive rats in vivo.

BACKGROUND: In order to identify a suitable rodent model for preclinical study of calcium antagonists, the pharmacokinetics and metabolism of one of the prototypes diltiazem (DTZ) in normotensive Sprague Dawley (SDR) was compared with Wistar Kyoto (WKY) rats and spontaneously hypertensive rats (SHR) following 5 mg/kg twice daily for five doses given by subcutaneous injection. METHODS: Pharmacokinetic data were analyzed by standard procedures assuming a one-compartment model with first-order input using Rstrips(®), and differences between the groups were considered significant when p<0.05. RESULTS: Plasma concentrations of DTZ were higher in the SHR than the normotensive SDR and WKY rats, although the differences did not reach statistical significance (p>0.05). Plasma concentrations of the active metabolites N-desmethyl DTZ (MA), deacetyl DTZ (M1) and deacetyl N-desmethyl DTZ (M2) were significantly higher in the SHR and WKY rats than the SDR, which was attributed to higher DTZ concentrations and also genetic factors. CONCLUSIONS: Although the differences were mainly quantitative and very small, the study has shown for the first time that the metabolism profiles of DTZ in SHR and WKY rats were closer to humans than SDR, and they may be more preferable rat models to study pharmacokinetic and metabolism studies of DTZ or similar agents.

Exercise improves hemodynamic profiles and increases red blood cell concentrations of purine nucleotides in a rodent model.

OBJECTIVE: To study the effect of exercise on hemodynamic profiles and red blood cell (RBC) concentrations of adenosine-5'-triphosphate (ATP). METHODS: Male Sprague-Dawley (SD) rats (n = 9) were exercised on a treadmill for 15 min at a speed of 10 m/min with a 5% gradient after an hour settling down in a restrainer. Blood samples were collected via an indwelling carotid artery catheter using a 'Stopping Solution' from each rat before, during and after exercise. Hemodynamic recordings were collected continuously throughout the experiment. Concentrations of ATP and other purine nucleotides in the RBCs were determined by a validated high-performance liquid chromatography (HPLC) assay. A control group (n = 12) was treated the same way except without the exercise. Data between groups were analyzed by analysis of variance (ANOVA) and Student's t-test, and differences were considered significant when p < 0.05. RESULTS: Exercise increased systolic blood pressure (SBP; 141±23 vs. 132±17 mmHg) and heart rate (HR; 420±33 vs. 397±41 bpm), but decreased diastolic blood pressure (DBP; 105±18 vs. 110±14 mmHg). This was followed by a postexercise condition when SBP, DBP, and HR were decreased for the remainder of the experiment. RBC concentrations of ATP and guanosine-5'-triphosphate (GTP) increased significantly during exercise and continued to increase for 5 hours postexercise (1.5±0.75 vs. 0.96 vs. 0.36 mM for ATP; and 0.14±0.061 vs. 0.058±0.030 mM) (p < 0.05). CONCLUSION: Exercise increased RBC ATP concentrations in a rodent model, which was correlated with the decrease in BP and HR postexercise.

Cladribine inhibits a diltiazem-induced increase in red blood cell purine nucleotide concentrations in a zebrafish model.

Minimizing drug interactions is paramount to improving the efficacy and tolerability of cancer therapy. The zebrafish represents an innovative cancer model due to highly conserved genetics and inherent capacity for high-throughput chemical screening. This pilot study extends the utility of the zebrafish to a preclinical model for pharmacodynamics by examining the interaction of the nucleoside analogue, cladribine with the calcium channel blocker, diltiazem. Cladribine (0.7-3.5 mM) and/or diltiazem (2.4 mM), was injected intraperitoneally into adult zebrafish and red blood cell (RBC) lysates were assayed by HPLC for levels of purine nucleotides (e.g. ATP), potential biomarkers of cardiovascular health. Diltiazem increased RBC ATP concentrations, which were inhibited by co-injection of cladribine. These results suggest a novel drug interaction and highlight the feasibility of the zebrafish as an in vivo model for pharmacodynamic studies.

Permeation of losartan across human respiratory epithelium: an in vitro study with Calu-3 cells.

The potential for nasal delivery of losartan, a drug with poor oral bioavailability, was investigated using Calu-3 cells. Epithelial permeation of the drug with or without dimethyl-beta-cyclodextrin (DM-beta-CD) and glycocholate was investigated. Possible transport mechanism of the compound and epithelial mucosal tolerance were screened. Reversibility of epithelial membrane perturbation was also investigated by measuring transepithelial electrical resistance (TEER) recovery over a 24-h period following drug formulation exposure. The permeability coefficient of losartan was 1.3 + or - 0.5 x 10(-6) cm s(-1). This flux was not significantly different from that of formulations containing DM-beta-CD (0.5 and 1.0%) or glycocholate (0.5%). However, the formulation with 1.0% glycocholate significantly increased losartan permeation 7-fold. Losartan flux across the cells was concentration-dependent. Serosal to mucosal permeation was significantly higher than mucosal to serosal permeation. Concentration-dependency, as well as polarity in transport indicated that the flux of the compound across Calu-3 cells was not limited to passive diffusion. Cells exposed to DM-beta-CD (0.5 and 1.0%) and glycocholate (0.5%) caused no significant change in TEER and mitochondrial dehydrogenase activity (MDH). The results of the study showed that losartan may be a suitable drug candidate for nasal delivery.

Pharmacokinetics and metabolism of diltiazem following multiple doses: comparing normotensive rat vs. hypertensive rat models in vivo.

In order to identify a suitable rodent model for preclinical study of calcium antagonists, pharmacokinetics and metabolism of diltiazem (DTZ) were compared in normotensive SD and hypertensive SHR rat models following multiple doses (5 mg/kg twice daily for 5 doses). Plasma concentrations of DTZ and its major metabolites appeared to be higher in the SHR than the SD rats, although the differences did not reach statistical significance (p > 0.05). The preliminary results suggest metabolism profile of DTZ in the SHR may be closer to humans than the SD rats and may be more preferred in pre-clinical drug development studies for DTZ.

Pharmacokinetics of cladribine in a rat model following subcutaneous and intra-arterial injections.

Male Sprague Dawley rats (n = 6-8 per group) weighing from 300-450 g were used for the study. Each rat received a single dose of cladribine (CdA) by ia (1 mg/kg) or s.c. (2 mg/kg) injection. Pharmacokinetic data were calculated by standard procedures assuming a 2-compartment open model following i.v. bolus using WinNonLin and Rstrips, and differences between the two modes of injections were considered significance when p < 0.05. The results showed that plasma concentrations of CdA decreased rapidly following a biphasic decline after both ia and s.c. administrations. The AUC and t1/2 beta after a single 1 mg/kg ia and 2 mg/kg s.c. injection of CdA were 0.66 +/- 0.34 vs 1.2 +/- 0.3 microg x h/ml and 3.5 +/- 2.1 vs 4.5 +/- 2.2 h, respectively (p > 0.05). The mean absolute bioavailability following the s.c. injection was close to 90%. The inter-subject variability of plasma concentrations of CdA was 35% and 150% following sc and ia injections, respectively. It is concluded that the rat is a reasonably good animal model to study the pharmacokinetics of CdA in plasma, and that sc injection may produce more favourable pharmacokinetic profiles than ia injection following a single dose.

Pharmacokinetics and metabolism of diltiazem in rats: comparing single vs repeated subcutaneous injections in vivo.

The objective of the study was to determine the effect of repeated administration on the pharmacokinetics and metabolism of diltiazem (DTZ) using an in vivo rat model. Male SD rats (n = 6-10 per group) weighing 350-450 g were used. Each rat received either a single 20 mg/kg dose of DTZ by subcutaneous (s.c.) injection or 5 mg/kg s.c. twice daily for five doses. Plasma concentrations of DTZ and its major metabolites were determined by HPLC for up to 8 h. Compared with the single dose, repeated administration resulted in higher dose normalized plasma concentrations of DTZ (AUC 26.4+/-14.2 vs 13.9+/-11.5 microg-h/ml), longer apparent half-life (t(1/2) = 12.5+/-14.6 vs 3.7+/-1.4 h) and lower systemic clearance (CL = 1.1+/-1.0 vs 2.9+/-2.7 l/h/kg). Higher dose normalized plasma concentrations, longer t(max), but shorter apparent t(1/2) of the major metabolites were observed following the repeated administration. The results also suggest that possible binding of DTZ may occur at the site of injection when administered subcutaneously in the higher dose.

Development and validation of a sensitive and specific HPLC assay of cladribine for pharmacokinetics studies in rats.

PURPOSE: To develop and validate a sensitive and specific HPLC assay for cladribine (CdA) in plasma for pharmacokinetic studies in rats. METHODS: CdA and the internal standard AZT were purchased from Sigma-Aldrich Chem. The HPLC system consisted of a Shimadzu LC-9A pump, a 3 im, 250 x 2.0 mm I.D. high speed C18 column (Jupitertrade mark), preceded by a 5 im 4 4 mm I.D. C18 guard column (Licrocarttrade mark), an Agilent Model 1050 UV-VIS detector and a 3395 Integrator. The mobile phase was made up of 0.01M KH2PO4 (pH 5): methanol: acetonitrile 90:5:5). The system was operated at ambient temperature with a flow rate of 0.3 mL/min, and UV wavelength at 265 nm, and an operating pressure of ca. 1.56 kpsi. Extraction of cladribine and AZT from plasma was achieved by solid phase extraction using 100 mg/mL C18 SPE columns Extra-septrade mark). The assay was validated for sensitivity, precision, specificity and application for pharmacokinetic study in rats. RESULTS: Under these conditions, the average retention times of CdA and AZT were 13.5 and 21 min, respectively, and recoveries were between 80 - 95%. Standard curve constructed from plasma standards was linear from 0.1 ug/mL to 1 ug/mL with regression coefficient (r2) 0.99 or greater. Sensitivity assessed by on column injection was < 1 ng. Using a 50-uL plasma sample size, the mean intra assay variations 0.1 ug/mL were 7%, and inter assay variations over a period of 3 months for 5 separate batches were less than 20%. The assay was used to study a single dose pharmacokinetic study of CdA in rats after a 2 mg/kg subcutaneous injection. CONCLUSION: The described HPLC assay has adequate sensitivity and specificity to study pharmacokinetics of CdA in rats, and could be adapted also to clinical pharmacokinetic studies.

Exercise hemodynamic and neurohormone responses as sensitive biomarkers for diltiazem in rats.

PURPOSE: To investigate the potential of exercise hemodyanamic and neurohormone variables as sensitive biomarkers for pre-clinical evaluation of diltiazem (DTZ). METHODS: Sprague Dawley (SD) rats were randomly divided into 3 groups (n = 6 - 8 each), and each group received DTZ 10 mg/kg twice daily for 5 doses or saline followed by a treadmill exercise protocol for 7 min with speed set at 7 m/min at 3 % grade. The 3rd group received saline but no exercise. RESULTS: Exercise increased SBP from 108 +/- 2 to 131 +/- 3 mmHg, and HR from 437 +/- 6 to 503 +/- 6 bpm, and plasma epinephrine concentrations from 2.0 +/- 0.6 to 5.8 +/- 1.7 ng/mL in control rats (p < 0.05 for all variables), but had no significant effect on DBP (81 +/- 5 vs 87 +/- 6 mmHg) and plasma norepinephrine concentrations (1.5 +/- 0.2 vs 3.9 +/- 0.4 ng/mL). The hemodynamic responses to exercise were significantly attenuated by DTZ (p < 0.05), but the effect on neurohormone response was minimal (p > 0.05). CONCLUSION: Exercise hemodynamic and neurohormone responses are sensitive biomarkers which could be used for safety and efficacy evaluation of DTZ and perhaps also other calcium antagonists in pre-clinical animal models.

Biomarker World Congress 2005.

This report covers some of the many excellent talks, and a selected number of posters, that were presented at this conference. It includes several emerging issues in biomarker development and the question of how biomarker science can drive targeted drug discovery and development and form a scientific basis for personalized medicine. Although relatively small, the meeting provided a good opportunity for business networking, particularly for those involved in the development and regulation of medical diagnostics and biopharmaceuticals.

DP-b99 (D-Pharm).

DP-b99 is a derivative of the calcium chelator BAPTA that is under development as a neuroprotectant for the potential treatment of stroke, head trauma and neurological damage associated with coronary artery bypass graft. By March 2003, phase II clinical trials in acute stroke and traumatic brain injury were ongoing.