Feasibility of 2–deoxy–2–[18F]fluoro–D–glucose– A85380–PET for imaging of human cardiac nicotinic acetylcholine receptors in vivo

Summary  Nicotinic acetylcholine receptors mediate the parasympathetic autonomic control of cardiac function. Aim of this study was the assessment of cardiac nicotinic acetylcholine receptor distribution with a novel (α₄β₂) nicotinic acetylcholine receptor PET ligand (2–deoxy–2– [¹⁸F]fluoro–D–glucose–A85380) in humans. Five healthy volunteers without cardiac disease and six patients with either Parkinson's disease or multiple system atrophy without additional overt cardiac disease were evaluated with 2–deoxy–2–[¹⁸F]fluoro–D–glucose–A85380 PET–imaging to assess the cardiac parasympathetic innervation and the putative impact of both disorders. 2–deoxy–2– [¹⁸F]fluoro–D–glucose–A85380 whole body PET–scans were performed on a Siemens PET/CT biograph^TM 75.4 min±6.7 after i.v. injection of 371.2±58.1 MBq. Average count rate density of left ventricle ROI's and a standard ROI in the right lung were measured within three consecutive slices of 10.0 mm thickness. Heart–to–lung ratios were calculated in each volunteer and patient. Tracer uptake in the left ventricle could be measured in all of the five volunteers and the six patients. Heart–to–lung ratios in the volunteer group were not different from patients suffering from Parkinson's disease or MSA (3.2 ± 0.5 vs 3.2 ± 0.8 and 2.96±0.7, mean ± SD), respectively. Human cardiac nicotinic acetylcholine receptors can be visualized and measured by 2–deoxy–2– [¹⁸F]fluoro–D–glucose–A85380 PET scans both in cardiac–healthy subjects and patients suffering from Parkinson's disease or multiple system atrophy. The heart– as well as the lung–tracer uptake was almost constant throughout all subjects leading to a good target–to–background ratio. These first results suggest no impact of either PD or MSA on cardiac nicotinic acetylcholine receptors. The Article “Feasibility of 2-deoxy-2-[¹⁸F]fluoro-D-glucose-A85380-PET for imaging of human cardiac nicotinic acetylcholine receptors in vivo” by J. Bucerius et al. (the online version can be found at has been published in issue 95 (2006):105–109. Unfortunately a wrong notation for the tracer was given. Instead of 2-deoxy-2-[¹⁸F]fluoro-D-glucose-A85380 it should be 2-[¹⁸F]-A85380. The publisher apologies for any inconvenience caused by this mistake.     

Feasibility of 2–deoxy–2–[18F]fluoro–D–glucose– A85380–PET for imaging of human cardiac nicotinic acetylcholine receptors in vivo

Summary Nicotinic acetylcholine receptors mediate the parasympathetic autonomic control of cardiac function. Aim of this study was the assessment of cardiac nicotinic acetylcholine receptor distribution with a novel (α₄β₂) nicotinic acetylcholine receptor PET ligand (2–deoxy–2– [¹⁸F]fluoro–D–glucose–A85380) in humans. Five healthy volunteers without cardiac disease and six patients with either Parkinson's disease or multiple system atrophy without additional overt cardiac disease were evaluated with 2–deoxy–2–[¹⁸F]fluoro–D–glucose–A85380 PET–imaging to assess the cardiac parasympathetic innervation and the putative impact of both disorders. 2–deoxy–2– [¹⁸F]fluoro–D–glucose–A85380 whole body PET–scans were performed on a Siemens PET/CT biograph^TM 75.4 min±6.7 after i.v. injection of 371.2±58.1 MBq. Average count rate density of left ventricle ROI's and a standard ROI in the right lung were measured within three consecutive slices of 10.0 mm thickness. Heart–to–lung ratios were calculated in each volunteer and patient. Tracer uptake in the left ventricle could be measured in all of the five volunteers and the six patients. Heart–to–lung ratios in the volunteer group were not different from patients suffering from Parkinson's disease or MSA (3.2 ± 0.5 vs 3.2 ± 0.8 and 2.96±0.7, mean ± SD), respectively. Human cardiac nicotinic acetylcholine receptors can be visualized and measured by 2–deoxy–2– [¹⁸F]fluoro–D–glucose–A85380 PET scans both in cardiac–healthy subjects and patients suffering from Parkinson's disease or multiple system atrophy. The heart– as well as the lung–tracer uptake was almost constant throughout all subjects leading to a good targetto– background ratio. These first results suggest no impact of either PD or MSA on cardiac nicotinic acetylcholine receptors. An erratum to this article is available at .     

Semiautomated Radiosynthesis and Biological Evaluation of [<Superscript>18</Superscript>F]FEAU: A Novel PET Imaging Agent for <Emphasis Type="Italic">HSV1-tk/sr39tk</Emphasis> Reporter Gene Expression

2′-Deoxy-2′-[¹⁸F]fluoro-5-ethyl-1-β-d-arabinofuranosyluracil ([¹⁸F]FEAU) is a promising radiolabeled nucleoside designed to monitor Herpes Simplex Virus Type 1 thymidine kinase (HSV1-tk) reporter gene expression with positron emission tomography (PET). However, the challenging radiosynthesis creates problems for being able to provide [¹⁸F]FEAU routinely. We have developed a routine method using a commercial GE TRACERlab FX-FN radiosynthesis module with customized equipment to provide [¹⁸F]FEAU. All radiochemical yields are decay corrected to end-of-bombardment and reported as means ± SD. Radiofluorination (33 ± 8%; n = 4), bromination (85 ± 8%; n = 4), coupling reaction (83 ± 6%; n = 4), base hydrolysis steps, and subsequent high-performance liquid chromatography purification afforded purified [¹⁸F]FEAU β-anomer in 5 ± 1% overall yield (n = 3 runs) after ~5.5 h and a β/α-anomer ratio of 7.4. Radiochemical/chemical purities and specific activity exceeded 99% and 1.3 Ci/μmol (48 GBq/μmol), respectively. In cell culture, [¹⁸F]FEAU showed significantly (P < 0.05) higher accumulation in C6 cells expressing HSV1-tk/sr39tk as compared to wild-type C6 cells. Furthermore, [¹⁸F]FEAU showed slightly higher accumulation than 9-[4-[¹⁸F]fluoro-3-(hydroxymethyl)butylguanine ([¹⁸F]FHBG) in cells expressing HSV1-tk (P < 0.05), whereas [¹⁸F]FHBG showed significantly higher (P < 0.05) accumulation than [¹⁸F]FEAU in HSV1-sr39tk-expressing cells. micro-PET imaging of mice carrying tumor xenografts of C6 cells stably expressing HSV1-tk or HSV1-sr39tk are consistent with the cell uptake results. The [¹⁸F]FEAU mouse images also showed very low gastrointestinal signal with predominant renal clearance as compared to [¹⁸F]FHBG. The routine radiosynthesis of [¹⁸F]FEAU was successfully semiautomated using a commercial module along with customized equipment to provide the β-anomer in modest yields. Although further studies are needed, early results also suggest [¹⁸F]FEAU is a promising PET radiotracer for monitoring HSV1-tk reporter gene expression.     

High-Yield,Automated Radiosynthesis of 2-(1-{6-[(2-[<Superscript>18</Superscript>F]Fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)malononitrile ([<Superscript>18</Superscript>F]FDDNP) Ready for Animal or Human Administration

The biomarker 2-(1-{6-[(2-[¹⁸F]fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)malononitrile ([¹⁸F]FDDNP) is used as a positron emission tomography (PET) imaging probe for Alzheimer’s disease and other neurodegenerative diseases. A high-yield and fully automated synthesis of [¹⁸F]FDDNP—along with the synthesis and characterization of non-radioactive FDDNP, a fluorescent probe derived from 2-(1,1-dicyanopropenyl-2)-6-dimethylaminonaphthalene (DDNP)—are reported. Radiofluorination of the tosyloxy precursor 2-{[6-(2,2-dicyano-1-methylvinyl)-2-naphthyl](methyl)amino}ethyl-4-methylbenzenesulfonate (DDNPTs) with K¹⁸F/Kryptofix 2.2.2. yielded chemically (>99%) and radiochemically (>99%) pure [¹⁸F]FDDNP in high radiochemical yields (40–60%; n> 120), with specific activities ranging from 4 to 8 Ci/μmol at the end of synthesis (90 minutes). Both remote, semiautomated and automated synthesis procedures are described. Either approach provides a reliable method for production of large quantities (110–170 mCi from 500 mCi of [¹⁸F]fluoride) of [¹⁸F]FDDNP allowing for multiple PET experiments in the same day or for distribution of the tracer from a single cyclotron facility to PET imaging centers at various geographical distances.     

Preclinical Acute Toxicity Studies and Dosimetry Estimates of the Novel Sigma-1 Receptor Radiotracer, [18F]SFE

[¹⁸F]1-(2-Fluoroethyl)-4-[(4-cyanophenoxy)methyl]piperidine ([¹⁸F]SFE) is a novel, selective, high-affinity sigma-1 receptor radioligand that has been preclinically well characterized in rodents. To support an investigational new drug (IND) application for the first evaluation of [¹⁸F]SFE in humans, single-organ and whole-body radiation adsorbed doses associated with [¹⁸F]SFE injection were estimated from rat distribution data. In addition, single- and multiple-dose toxicity studies were conducted in rabbits and in dogs. Multiple-dose toxicity studies in rabbits and single-dose toxicity studies in beagles suggest at least a 100-fold safety margin for humans studies at a mass dose limit of 4.0 μg per intravenous injection, based on the combined no observable adverse effect levels (NOAEL, mg/m²) measured in these species. Radiation dosimetry estimates obtained from rat biodistribution analyses of [¹⁸F]SFE suggest that most tissues would receive about 0.010–0.020 mGy/MBq, while the adrenal glands, brain, bone, liver, lungs, and spleen would receive slightly higher doses (0.024–0.044 mGy/MBq). The adrenal glands were identified as the critical organ, because they received the highest adsorbed radiation dose. The total exposure resulting from a 5 mCi administration of [¹⁸F]SFE is well below the FDA-defined limits for yearly cumulative and per-study exposures to research participants. These combined results support the expectation that [¹⁸F]SFE will be safe for use in human positron emission tomography (PET) imaging studies with the administration of 5 mCi and a mass dose equal to or less than 4.0 μg SFE per injection.     

Limitations of Small Animal PET Imaging with [18F]FDDNP and FDG for Quantitative Studies in a Transgenic Mouse Model of Alzheimer’s Disease

Purpose  We evaluated the usefulness of small animal brain positron emission tomography (PET) imaging with the amyloid-beta (Aβ) probe 2-(1-{6-[(2-[¹⁸F]fluoroethyl)(methyl)amino]-2-naphthyl}ethylidene)malonitrile ([¹⁸F]FDDNP) and with 2-deoxy-2-[F-18]fluoro-d-glucose (FDG) for detection and quantification of pathological changes occurring in a transgenic mouse model of Alzheimer’s disease (Tg2576 mice). Procedures  [¹⁸F]FDDNP (n = 6) and FDG-PET scans (n = 3) were recorded in Tg2576 mice (age 13–15 months) and age-matched wild-type litter mates. Brain volumes of interest were defined by co-registration of PET images with a 3D MOBY digital mouse phantom. Regional [¹⁸F]FDDNP retention in mouse brain was quantified in terms of the relative distribution volume (DVR) using Logan’s graphical analysis with cerebellum as a reference region. Results  Except for a lower maximum brain uptake of radioactivity in transgenic animals, the regional brain kinetics as well as DVR values of [¹⁸F]FDDNP appeared to be similar in both groups of animals. Also for FDG, regional radioactivity retention was almost identical in the brains of transgenic and control animals. Conclusions  We could not detect regionally increased [¹⁸F]FDDNP binding and regionally decreased FDG binding in the brains of Tg2576 transgenic versus wild-type mice. However, small group differences in signal might have been masked by inter-animal variability. In addition, technical limitations of the applied method (partial volume effect, spatial resolution) for measurements in such small organs as mouse brain have to be taken into consideration.     

Hemodynamic responses to successful weaning from mechanical ventilation after cardiovascular surgery

Weaning from mechanical ventilation is usually associated with an increase in oxygen consumption (VO₂), which may stress the cardiovascular system. We studied relative changes in the cardiac index and oxygen extraction ratio (EO₂) during successful weaning in patients after cardiac surgery (n = 52), cardiac transplantation (n = 17), or abdominal aortic surgery (n = 11). Cardiac index was determined by the thermodilution technique and arterial and mixed venous blood gases were obtained before and 30 min after the start of weaning through a T-piece. The cardiovascular changes were evaluated in 42 patients in whom VO₂ (calculated by Fick's equation) increased by more than 10 %. Cardiac index increased more after abdominal aortic surgery (from 3.27 ± 0.77 to 4.44 ± 0.58 l min^–1 m^–2, p < 0.01) than after cardiac surgery (from 2.53 ± 0.59 to 2.87 ± 0.46 l min^–1 m^–2, p < 0.01) or cardiac transplantation (from 2.99 ± 0.64 to 3.33 ± 0.74 l min^–1 m^–2, p < 0.05). EO₂ remained stable in patients after aortic surgery (from 25.9 ± 7.1 to 25.2 ± 5.6 %, NS) but increased slightly after cardiac surgery (from 33.3 ± 6.1 to 37.3 ± 6.4 %, NS) and significantly after cardiac transplantation (from 25.8 ± 4.1 to 28.2 ± 4.0 %, p < 0.05). Hence the cardiovascular response to weaning from mechanical ventilation may vary according to the type of surgery. Received: 14 June 1999/Final revision received: 18 April 2000/Accepted: 26 April 2000     

Pharmacological and toxicological evaluation of 2-fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2-F-A-85380), a ligand for imaging cerebral nicotinic acetylcholine receptors with positron emission tomography

2-[(18)F]fluoro-3-(2(S)-azetidinylmethoxy)pyridine (2-[(18)F]F-A-85380), a positron emission tomography (PET) radioligand for neuronal alpha4beta2(*) nicotinic acetylcholine receptors, was evaluated for its pharmacology and safety. In the Ames test for mutagenicity, 2-F-A-85380 was without effect in five bacterial strains. No evidence of gross pathology or histopathological changes occurred in either 2-day acute (0.4-4000 nmol/kg i.v.) or 14-day expanded acute (40-4000 nmol/kg i.v.) toxicity studies in mice. Similarly, hematology and serum chemistry values in rhesus monkeys administered 60 nmol/kg i.v. were not affected over 14 days. Like nicotine, 2-F-A-85380 produced convulsions in mice at very high doses. The ED(50) value of 2-F-A-85380 for eliciting tonic-clonic convulsions (5.0 micromol/kg i.v.) was nearly 4 times greater than that of nicotine (ED(50) = 1.4 micromol/kg i.v.). Lower doses of 2-F-A-85380 (30-300 nmol/kg i.v.) and nicotine (20-400 nmol/kg i.v.) increased systolic and diastolic blood pressure, heart rate, and cardiac contractility in rats. Notably, the PR, QRS, or QTc intervals of the rat electrocardiogram were unaffected by either drug. Dosimetry studies indicated that the urinary bladder wall was the critical organ and total radiation exposure was within acceptable limits. Estimated doses of 2-F-A-85380 required to elevate blood pressure and heart rate by 10% ranged from 40 to 58 nmol/kg i.v. Nevertheless, the estimated radiopharmaceutically relevant dose of [(18)F]2-F-A-8380 required for initial PET imaging studies, 10 pmol/kg, is less than 1/4000th of the doses calculated (40-58 nmol/kg i.v.) to elevate blood pressure and heart rate by 10% in humans and should elicit no clinically significant effects and have acceptable dosimetry.     

Tyrosine Kinase Inhibitors Reduce Glucose Uptake by Binding to an Exofacial Site on hGLUT‐1: Influence on 18F‐FDG PET Uptake

Positron emission tomography (PET) using 2‐deoxy‐2‐[¹⁸F]fluoro‐d‐glucose ([¹⁸F]FDG), a marker of energy metabolism and cell proliferation, is routinely used in the clinic to assess patient response to chemotherapy and to monitor tumor growth. Treatment with some tyrosine kinase inhibitors (TKIs) causes changes in blood glucose levels in both nondiabetic and diabetic patients. We evaluated the interaction of several classes of TKIs with human glucose transporter‐1 (hGLUT‐1) in FaDu and GIST‐1 cells by measuring [³H]2‐deoxy‐d‐glucose ([³H]2‐DG) and [³H]FDG uptake. Uptake of both was inhibited to varying extents by the TKIs, and representative TKIs from each class showed competitive inhibition of [³H]2‐DG uptake. In GIST‐1 cells, [³H]FDG uptake inhibition by temsirolimus and nilotinib was irreversible, whereas inhibition by imatinib, gefitinib, and pazopanib was reversible. Molecular modeling studies showed that TKIs form multiple hydrogen bonds with polar residues of the sugar binding site (i.e., Q161, Q282, Q283, N288, N317, and W388), and van der Waals interactions with the H‐pocket site. Our results showed interaction of TKIs with amino acid residues at the glucose binding site to inhibit glucose uptake by hGLUT‐1. We hypothesize that inhibition of hGLUT‐1 by TKIs could alter glucose levels in patients treated with TKIs, leading to hypoglycemia and fatigue, although further studies are required to evaluate roles of other SLC2 and SLC5 members. In addition, TKIs could affect tumor [¹⁸F]FDG uptake, increasingly used as a marker of tumor response. The hGLUT‐1 inhibition by TKIs may have implications for routine [¹⁸F]FDG‐PET monitoring of tumor response in patients.

Study Highlights

• WHAT IS THE CURRENT KNOWLEDGE ON THE TOPIC?

☑ Human glucose transporter‐1 transports glucose and 18‐flurodexoyglucose (FDG) into cells where it gets phosphorylated and trapped. A retrospective study of blood glucose concentrations in diabetic and nondiabetic patients with cancer treated with dasatinib, imatinib, sorafenib, or sunitinib showed a statistically significant decrease in blood glucose levels.

• WHAT QUESTION DID THIS STUDY ADDRESS?

☑ Inhibition of glucose uptake was examined and tyrosine kinase inhibitors (TKIs) inhibited [³H]2‐DG and [³H]FDG uptake to varying extents and competitively. Molecular modeling studies confirmed TKI interaction with amino acid residues at glucose binding sites.

• WHAT DOES THIS STUDY ADD TO OUR KNOWLEDGE?

☑ We hypothesized that TKI inhibition of glucose uptake might lead to changes in tissue and blood glucose levels and might explain the hypo/hyperglycemia and fatigue observed in TKI‐treated patients.

• HOW MIGHT THIS CHANGE CLINICAL PHARMACOLOGY OR TRANSLATIONAL SCIENCE?

☑ Because FDG uptake is increasingly used as a marker of tumor response, these results indicate careful evaluation of [¹⁸F]FDG‐positron emission tomography tumor response monitoring during TKI treatment, and in clinical trials validating new TKI effectiveness.

Clinical decision making involves cancer treatment response monitoring to prevent ineffective therapy and resulting side effects. Positron emission tomography (PET) is used for monitoring treatment effectiveness. PET tracers 2‐deoxy‐2‐[¹⁸F]fluoro‐d‐glucose ([¹⁸F]FDG) and 3′‐deoxy‐3′‐[¹⁸F]fluorothymidine have been used for noninvasive detection of tumor energy metabolism and cell proliferation, respectively. The glucose analog [¹⁸F]‐FDG is the most widely used PET tracer for tumor staging and diagnosis, treatment response, and routine treatment monitoring. ¹ Human glucose transporter‐1 (hGLUT‐1) transports FDG, like glucose, into cells where it subsequently gets phosphorylated and trapped. ² Many small molecule tyrosine kinase inhibitors (TKIs), such as erlotinib, gefitinib and vandetanib, and lapatinib, targeted against epidermal growth factor receptor (EGFR), vascular endothelial growth factor receptor (VEGFR) and EGFR, and EGFR and Her‐2, respectively, have been developed. Multitargeted TKIs include sorafenib, axitinib, pazopanib, and sunitinib. TKIs targeting intracellular BCR‐ABL kinase of chronic myeloid leukemia include bosutinib, dasatinib, imatinib, nilotinib, and ponatinib. Multiple TKIs are used successfully for treatment of renal cell carcinoma, leukemia, gastrointestinal tumors, lung cancer, and several other malignancies. A retrospective study of blood glucose concentrations in diabetic and nondiabetic patients with cancer treated with dasatinib, imatinib, sorafenib, or sunitinib showed statistically significant decrease in blood glucose levels. ³ Many preclinical studies have investigated use of [¹⁸F]FDG PET imaging for TKI treatment monitoring, and some studies showed early changes in [¹⁸F]FDG levels while others did not. ⁴ Depending on the TKI used, accumulating evidence show either increased or decreased glucose levels in patients during TKI therapy, although dasatinib, erlotinib, sunitinib, and sorafenib were shown to lower glucose levels and improve glycemic control. ⁵ PET tracer uptake is complex and depends on tumor type, targeted kinase, and tracer uptake by tumors. In general, higher standardized uptake values have been correlated with poorer outcomes. ⁶ Solute carrier (SLC) transporters for sugars include SLC2 and SLC5 families, which regulate cellular concentrations of sugars. ⁷ , ⁸ hGLUT‐1, a widely distributed member of the facilitative SLC2 family, transports glucose down its concentration gradient. ⁹ XylE, a structurally characterized xylose transporter of E. coli, shows 65% sequence identity and 88% sequence similarity with hGLUT‐1. ¹⁰ Residues important for binding of d‐glucose (DG) in the crystal structure of XylE (although XylE does not transport dg) are conserved in hGLUT‐1 with the exception of Gln415, which is Asn411 in hGLUT‐1. Gln‐282 contributes to sugar binding in all conformations of hGLUT‐1 via hydrogen bonding. ¹¹ The sugar‐binding site is located at the interface of N‐terminal and C‐terminal domains. An earlier study showed competitive inhibition of glucose transport activity of hGLUT‐1 by flavones and isoflavones. ¹² WZB117, a polyphenol compound, inhibits erythrocyte 3‐O‐methylglucose uptake competitively by binding at the exofacial binding site. ¹³ We examined whether hGLUT‐1 activity is inhibited by TKIs targeted against EGFR, VEGFR, Her‐2, BCR‐ABL, MTK, MEK, and mTOR using [³H]2‐deoxy‐d‐glucose ([³H]2‐DG) and [³H]FDG uptake inhibition experiments in cultured FaDu and GIST‐1 cells to understand underlying mechanisms of TKI‐induced changes in glucose levels of TKI‐treated patients. TKI inhibition of hGLUT‐1 was tested further in competition experiments using one representative TKI from BCR‐ABL, EGFR, MTK, and mTOR TKIs. Reversibility of TKI inhibition of [³H]FDG uptake after removing TKIs was examined in GIST‐1 cells. Further evidence for TKI interactions with hGLUT‐1 glucose binding site was obtained by molecular modeling using three models of outward‐facing partly occluded conformation (models 1–3), one model of inward‐open conformation (model 4), and one model of fully open outward‐facing conformation (model 5).     

Characterisation of left ventricular relaxation in the isolated guinea pig heart

The time constant of left ventricular pressure fall, τ, has frequently been used as a measure of myocardial relaxation in the blood-perfused, ejecting heart. The aim of the present study was to characterise τ in relation to β-adrenergic activation, coronary perfusion pressure and flow as well as cardiac oxygen supply and demand in the isolated, isovolumically beating heart. Therefore, τ was analysed from digitised left ventricular pressure data in a total of 23 guinea pig hearts perfused with saline at constant pressure (60 cmH₂O). The coronary venous adenosine concentration ([ADO]) served as an index of myocardial oxygenation. Isoprenaline (0.4–3.2 nmol l⁻¹) decreased and propranolol (3–9 μmol l⁻¹) increased τ dose-dependently (linear regression τ vs lg ([isoprenaline]),r=0.74; τ vs. lg([propranolol]),r=0.66, bothP<0.05). During graded reductions in cardiac oxygen supply from 96.1±12.6(SEM) to 44.4±4.4 μl min⁻¹ g⁻¹, τ was prolonged from 61.5±12.7 to 109.9±22.6 ms while left ventricular developed pressure (LVDP) decreased from 90.7±7.2 to 40.7±5.1 mmHg. In parallel, [ADO] increased from 23.7±9.1 to 58.0±19.1 pmol ml⁻¹ (P<0.05). Increasing oxygen supply to 165.4±32.4 μl min⁻¹ g⁻¹ augmented LVDP to 102.7±7.3 mmHg but did not change τ or [ADO]. There was a dual response of τ to changes in cardiac oxygen supply or demand. As long as oxygen supply and demand matched, τ remained constant. However, when the oxygen supply was less than 100 μl min⁻¹g⁻¹, left ventricular relaxation was prolonged in parallel to the reduction in oxygen supply. In addition, a close relationship was observed between [ADO] as an indicator of myocardial oxygenation and τ (Spearman correlation,r=0.99,P<0.005). We conclude that the time constant of left ventricular pressure fall, τ, sensitively reflects myocardial relaxation in the isolated, isovolumically beating guinea pig heart. Moreover, in this model left ventricular relaxation is not influenced by alterations in coronary perfusion pressure or flow as long as cardiac oxygen demand is matched by an adequate supply. Rather, relaxation is strictly coupled to myocardial oxygenation as reflected by coronary venous adenosine concentrations.