Intracellular pH (pHi) impacts many cellular mechanisms including cellular metabolism, gene expression, cell volume regulation, cell survival and proliferation. Most cells use two general pHi regulatory mechanisms: HCO3-/Cl- antiporters (AE, Slc4a family) to reduce internal alkaline load, and Na+/H+ exchangers (NHE, Slc9a family) that protect cells from acidosis. Previous studies with preimplantation (PI) embryos have shown robust activity of HCO3-/Cl- exchanger in all stages of development. It was also determined that inhibition of this exchange with the stilbene AE inhibitor 4,4’-diisothiocyanostilbene-2,2’-disulfonic acid (DIDS) was detrimental to embryo development from the 2‐cell stage to blastocyst when cultured at high external pH. In this study I investigated which of the five known plasma membrane NHE isoforms was present and active within mouse PI embryos and their role as pHi regulators throughout preimplantation embryo development. In mouse oocytes and preimplantation embryos, mRNAs were detected encoding NHE1 (SLC9A1), NHE3 (SLC9A3), and NHE4 (SLC9A4), with higher mRNA levels for each in fully-grown oocytes through one-cell stage embryos and then generally lower levels after the two-cell stage. No transcripts for NHE2 (SLC9A2) or NHE5 (SLC9A5) were detected. Measurements of intracellular pH during recovery from acidosis, induced by transient ammonium pulse, suggested that recovery occurred and was mediated by NHE activity at all preimplantation stages assessed (one-cell, two-cell, eight-cell and morula). This recovery was inhibited by 1 mM amiloride, a general NHE inhibitor. The observed residual recovery was attributed to passive passage of protons across the membrane, rather than the activity of NHE4 (an amiloride-resistant isoform), since no further decrease in recovery rates from acidosis was observed upon amiloride increase to 5 mM. Furthermore, recovery from acidosis at each stage was entirely inhibited by cariporide, which is very highly selective for NHE1. In contrast, the moderately NHE3-selective inhibitor S3226 did not preferentially block recovery, nor did adding S3226 increase inhibition over that achieved with cariporide alone, indicating that NHE3 did not play a functional role in pHi regulation at any stage assessed. Another regulator of intracellular pH against acidosis, previously reported to be active in oocytes and 1-cell embryos, the sodium-dependent bicarbonate/chloride exchanger (NDBCE; SLC4A8), had low or absent activity in two-cell embryos. This indicated that NHE1 is likely the only significant regulator of pHi in preimplantation mouse embryos, at least after the 1-cell stage. Culturing embryos from the one-cell or two-cell stages in acidotic medium inhibited their development, as assessed by development to the blastocyst stage and cell lineage allocation. However, inhibition of NHE1 with cariporide, NDBCE with DIDS, or both together did not further decrease embryo development to the blastocyst stage more extensively under conditions of chronic acidosis than at normal pH. This suggests that mouse PI embryos have a restricted ability to counteract chronic acidosis by means of pHi regulatory mechanisms, despite clearly being able to recover from acute acidosis via NHE1 activity.

Brain death has adverse effects on the organ donor, increasing organ dysfunction and affecting transplantation outcomes. It can also render organs unsuitable for transplantation. Another determinant of organ quality is ischaemia-reperfusion injury, which limits ischaemic storage time for hearts to six hours. The aim of this thesis was to investigate the effectiveness of hormone resuscitation (HR) of the donor to ameliorate the effects of brain death. Another aim was to develop a donor management and organ preservation strategy to ameliorate the effects of ischaemia-reperfusion injury on the heart, thereby extending ischaemic preservation times. A porcine model of the brain-dead multi-organ donor with orthotopic cardiac transplantation was utilised. Donor HR was shown to improve cardiac contractility and haemodynamics, thereby reducing inotrope requirements. A follow-up study investigating the effects of three different donor management protocols demonstrated that donor haemodynamics, renal arterial flow and creatinine clearance were superior in HR animals compared with animals treated with noradrenaline or intravenous fluid alone. Noradrenaline was associated with a significant deterioration in pulmonary function (PaO2 and alveolar-arterial oxygen gradient) and a decline in donor pH. HR was not associated with any detrimental effects on the lungs, liver or pancreas compared with the other two groups. Preservation strategies incorporating glyceryl trinitrate (GTN) and cariporide, a Na+-H+ exchange inhibitor, were investigated to safely extend cardiac ischaemic preservation times. Pre-treatment with intravenous cariporide prior to heart explantation (donor) and reperfusion of the transplanted heart (recipient) was shown to effectively extend ischaemic time to 14 hours, evidenced by weaning off cardiopulmonary bypass. GTN and cariporide-supplemented Celsior, used as a cardioplegic/storage solution, was also effective in extending preservation time to 14 hours, with superior cardiac contractility compared with cariporide pre-treated hearts. Both treatments also ameliorated reperfusion injury, stabilising haemodynamics for up to three hours post-bypass. This thesis has demonstrated the effectiveness of HR to ameliorate the negative effects of donor brain death. It also provides evidence that combined GTN and cariporide-supplemented Celsior improves long-term preservation of the donor heart. These strategies offer the potential to increase the proportion of transplantable organs, to improve donor organ quality, and thereby improve transplantation outcomes.

Die Beurteilung der Wirkung von Cariporide auf dieEreignis-Wahrscheinlichkeit eines Herzinfarktes oder des Todes imRahmen einer Bypass-Operation ist Gegenstand der Arbeit. DasNHE-Austauschersystem in der Herzmuskelzelle induziert den unterIschämie durch den intrazellulären Protonenüberschusshervorgerufenen Na(+)- und Ca(2+)-Einstrom. Cariporide ist einNHE-Inhibitor, der den unter Ischämie durch die Ca(2+)-Überladunginduzierten Herzmuskelzelltod verzögern soll. In einer kombinierten Phase-II/III-Studie (GUARDIAN, n=11590) war derEinfluss verschiedener intravenöser Dosen von Cariporide auf dieHäufigkeit von Herzinfarkt oder Tod in ACS/NQMI, PTCA undCABG-Patientenkollektiven untersucht worden. Nur die höchstdosierteCABG-Gruppe zeigte eine signifikante Reduktion der Ereignisrate um24,7% (p=0,027) gegenüber Placebo. Diese schwacheDosis-Wirkungs-Beziehung konnte durch eine pharmakokinetische undpharmakodynamische Populationsanalyse in eineKonzentrations-Wirkungs-Beziehung überführt werden. Zur Entwicklungdes Populationsmodells waren verschiedene Submodelle notwendig: 1. Modell für den Zeitverlauf der Ereignisrate: Durch Kombination zweier Weibull-Verteilungen ist es möglich, die beobachteten Daten als Überlebenszeitfunktion nach CABG zu beschreiben. Ein akutes, unmittelbar auf die CABG-Operation zurückzuführendes Risiko wird hier von einem chronischen Risiko unterschieden. 2. Pharmakokinetisches Modell: Ein multiexponentielles populationspharmakokinetisches Modell ist notwendig zur Beschreibung der PK nach iv-Applikation von Cariporide bei Probanden und Patienten. 3. Pharmakodynamisches Modell: Über ein empirisches logistisches Modell wird die Reduktion des akuten Risikos mit der mittleren Cariporide Plasmakonzentration unter der Bypass-Operation verknüpft. In einer Substudie der GUARDIAN-Hauptstudie konnte daspopulationspharmakokinetische Modell aus der früherenPhase-I-Entwicklung mit Probanden für die Patienten validiert werden.Die mit Hilfe der individüllen Dosierung, der demographischen Datenund dem Populationsmodell für die Periode mit dem höchsten Risikowährend der CABG-Operation vorhergesagten mittlerenPlasmakonzentrationen flossen in die Analyse derKonzentrations-Zeit-Abhängigkeit der Ereignis-Wahrscheinlichkeit ein. Eine untere Schwellenkonzentration (0,5mg/l), unterhalb der mitkeinem Effekt zu rechnen ist, wurde bestimmt. Die Daten erlaubten dieSchätzung des maximalen Effekts nur unzureichend. Die maximaleRisikoreduktion von 60% wurde mit einem Konfidenzintervall von29% bis 100% geschätzt. Unter Einsatz einer linearen Näherungdes Hill-Modells wurde eine obere Schwellenkonzentration bei 0,9mg/lbestimmt. Nur 37% aller Patienten der 80mg-Dosisgruppe erreichtenmittlere Konzentrationen oberhalb der unteren Schwellenkonzentration,in der 120mg-Dosisgruppe waren es immerhin schon 75% allerPatienten. Die Infusion von 120mg Cariporide über eine Stunde gefolgt voneiner Erhaltungsdosis von 20mg/h für weitere 47 Stunden sollte bei95% der Patienten während der CABG-Operation zu mittlerenKonzentrationen über der minimal effektiven Konzentration von0,5mg/l führen. Eine auf diese Weise mittels Simulationenoptimierte Dosierungsregel sollte während der CABG-Operation zu einemerhöhten Schutz der Patienten gegen die Folgen ischämischerEreignisse führen. Eine weitere Erhöhung der Erhaltungsdosis aufbis zu 40mg/h mit einer entsprechenden Anpassung der Initialdosissollte 95% der Patienten sogar über die bisher nur unsicher zubestimmende obere Grenzkonzentration von 0,9mg/l bringen. Solltenkeine dosislimitierenden Nebenwirkungen auftreten, kann dieseErhöhung sowohl der Initialdosis als auch der Erhaltungsdosis zueiner weiteren Verbesserung während der Risikoperiode führen undeinen weiteren potentiellen klinischen Vorteil für Cariporideerbringen. / Subject of this analysis is the assessment of the effect of cariporideon the event probability of a myocardial infarction (MI) or death inthe scope of a coronary artery bypass graft. Thesodium-hydrogen-exchange system (NHE) in the myocardial cell inducesthe sodium and calcium influx caused by an ischaemia induced hydrogenoverload. Cariporide is a NHE-inhibitor which is seen to be delayingthe necrosis of myocardial cells caused by the ischaemia inducedcalcium influx. The influence of different intravenous doses of cariporide on thefrequency of MI and death in ACS/NQMI, PTCA, and CABG patients hadbeen investigated in a combined phase II/III trial (GUARDIAN,n=11590). Only the highest dosed CABG-subgroup showed a significantreduction of the event-rate compared to placebo of 24.7% (p=0.027).This weak dose-effect-relationship could be translated into aconcentration-effect relationship by using a populationpharmacokinetic/pharmacodynamic (PK/PD) analysis. To develop thispopulation model a series of sub-models were established: 1) Model for the time-to-event progression: using a combination of two Weibull-distributions, it was possible to describe the observed data following the CABG procedure by means of a survival-function. An acute risk, likely to be related to the CABG-procedure could be discriminated from a chronic risk. 2) Pharmacokinetic model: a multi-exponential population PK model was necessary to describe the PK after iv-application of cariporide in volunteers as well patients. 3) Pharmacodynamic model: using an empirical logistic model the reduction of the acute risk was linked to the cariporide plasma-concentrations. In a sub-study of the GUARDIAN-main study, the population PK model ofthe phase I development in volunteers had been be validated. Usingthe individual dosing, the individual demographic information and thepopulation PK model mean concentrations were calculated for the periodof the highest risk during the CABG procedure. Those concentrationswere then introduced into the analysis of the concentration timedependency of the event probability. A lower threshold concentration (0.5mg/l) was estimated beneath thatno effect would be expected. The data permitted the estimation of themaximum effect only insufficiently. A maximum risk reduction of 60%was estimated with a confidence interval from 29% to 100%. Using thelinear approximation of the Hill-model an upper thresholdconcentration of 0.9mg/l could be determined. Mean concentrationsunder risk were reached above the lower threshold concentration inonly 37% of all patients in the 80mg dose group, whereas in the 120mgdose-group already 75% of the patients exceeded the lower thresholdconcentration. The infusion of 120mg cariporide for an hour followed by a maintenancedose of 20mg/h for further 47 hours should maintain in 95% of thepatients during the CABG-procedure mean concentrations above theminimal effective concentration of 0.5mg/l. A dose regimen optimisedin this sense by means of simulations should lead to an increasedprotection against ischemic events during and after theCABG-operation. A further increase of the maintenance dose up to40mg/h with a corresponding adaptation of the initial dose shouldshift at least 95% of the patients above the so far impreciseestimated upper threshold concentration of 0.9mg. If no dose limitingside-effects occur, this increase of both the initial dose and themaintenance dose may lead to a further improvement during the riskperiod and may result in a further potential clinical advantage forcariporide.

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