Apelin is an endogenous peptide with physiological actions in the cardiovascular system. Apelin can modulate vasomotor tone and is a potent endogenous inotrope. In a series of clinical studies, we have shown that apelin causes peripheral and coronary vasodilatation and increased cardiac contractility. We wish to study and compare the cardiovascular effects of subcutaneous versus intravenous apelin. We specifically wish to determine how different methods of apelin administration affect cardiac output and peripheral vascular resistance in healthy volunteers

Study Protocols Groups of 5 subjects will be assigned to receive one of three protocols.

Protocol 1:

Five healthy volunteers will be asked to attend the clinical research facility on a total of 4 occasions as per the protocol below.

Visits 1 and 2 will occur over two consecutive days. Participants will attend at 07.30 and be given a light breakfast. An intravenous sampling cannula will be inserted into the antecubital vein of one arm. Blood samples (5 mL) will be taken immediately prior to subcutaneous injection (t=0; 08.00) of 1 mg (Pyr1)apelin-13 and at 5, 10, 15, 20, 25, 30, 45, 60, 90, 120, 180, 240, 300, 360, 480 and 600 min after injection. Subjects will then be discharged from the facility and reattend the following day at 08.00 for a single venous sample. Approximately 85 mL of blood will be sampled in total over this time.

Visits 3 and 4 will also occur over two consecutive days, at least one week after visit 2. Subjects will attend at 07.30 and be given a light breakfast. An intravenous sampling cannula will be inserted into the antecubital vein of one arm. Blood samples (5 mL) will be taken immediately prior to an intravenous bolus infusion (t=0; 08.00) of 1 mg (Pyr1)apelin-13 over 15 min and at 5, 10, 15, 20, 25, 30, 45, 60, 90, 120, 180, 240, 300, 360, 480 and 600 min after injection. Subjects will then be discharged from the facility and reattend the following day at 08.00 for a single venous sample.

Protocol 2:

Five healthy volunteers will be asked to attend the clinical research facility on a total of 4 occasions as per the protocol below.

Visits 1 and 2 will occur over two consecutive days. Participants will attend at 07.30 and be given a light breakfast. An intravenous sampling cannula will be inserted into the antecubital vein of one arm. Blood samples (5 mL) will be taken immediately prior to subcutaneous injection (t=0; 08.00) of 5 mg (Pyr1)apelin-13 and at 5, 10, 15, 20, 25, 30, 45, 60, 90, 120, 180, 240, 300, 360, 480 and 600 min after injection. Subjects will then be discharged from the facility and reattend the following day at 08.00 for a single venous sample. Approximately 85 mL of blood will be sampled in total over this time.

Visits 3 and 4 will also occur over two consecutive days, at least one week after visit 2. Subjects will attend at 07.30 and be given a light breakfast. An intravenous sampling cannula will be inserted into the antecubital vein of one arm. Blood samples (5 mL) will be taken immediately prior to an intravenous bolus infusion (t=0; 08.00) of 5 mg (Pyr1)apelin-13 over 15 min and at 5, 10, 15, 20, 25, 30, 45, 60, 90, 120, 180, 240, 300, 360, 480 and 600 min after injection. Subjects will then be discharged from the facility and reattend the following day at 08.00 for a single venous sample.

Protocol 3:

Five healthy volunteers will be asked to attend the clinical research facility for 2 consecutive days. Subjects will attend at 07.30 and be given a light breakfast. An intravenous sampling cannula will be inserted into the antecubital vein of one arm. Blood samples (5 mL) will be taken immediately prior to commencing a 24-h subcutaneous infusion of (t=0; 08.00) of 10 mg (Pyr1)apelin-13 dissolved in 10 mL water for injection. Further venous sampling will take place at 5, 10, 15, 20, 25, 30, 45, 60, 90, 120, 180, 240, 300, 360, 480 and 600 min after injection. Subjects will then be discharged from the facility and reattend the following day at 08.00 for a single venous sample.

Measurements

Thoracic Bioimpedance Cardiography By virtue of changes in transthoracic electrical impedance during cardiac ejection, thoracic bioimpedance cardiography allows the non-invasive assessment of cardiac stroke volume and the calculation of cardiac output and cardiac index. After skin preparation, four pairs of low-contact impedance 'sensing' and 'current injecting' electrodes will be attached to the patient and connected to an impedance cardiograph. This technique has been applied widely and compares favourably with both invasive and other non-invasive (echocardiographic) measures of cardiac output. These variables will therefore be recorded at regular intervals throughout the study in all 3 protocols. Heart rate and blood pressure will also be monitored at regular intervals throughout each study using a semi-automated oscillometric sphygmomanometer (Omron HEM-705CP, Omron, Matsusaka, Japan). Mean arterial pressure (MAP) will be calculated as diastolic blood pressure plus a third of the pulse pressure.

Assays Blood samples (5 mL) will be collected before and at the end of each drug infusion into ethylene diamine tetraacetic acid (EDTA), centrifuged and plasma frozen in three 1-mL aliquots to be stored at -80 °C until assay. Plasma concentrations of apelin will be measured by collaborators at Bristol Myers Squibb, Princeton, USA.

Methods of Statistical Analysis

Outcome data will be analysed where appropriate, by analysis of variance (ANOVA) with repeated measures, regression analysis, and paired and unpaired Student's t-test. Statistical significance will be taken at the 5% level.

Inclusion Criteria:

• Healthy males or females >18 years old
• Willing and able to complete an informed consent form

Exclusion Criteria:

• Unable to give informed consent
• Pregnant or lactating at screening
• Use of any investigation product or device within 30 days prior to study.