Comparison of strand displacement and ligase chain amplification for detection of Chlamydia trachomatis infection in urogenital specimens.

Two amplification tests for the diagnosis of Chlamydia trachomatis infection, namely the ligase chain reaction (LCx) and the strand displacement assay (ProbeTec), were compared using samples from 1183 patients at sexually transmitted disease clinics. The overall prevalence of positive results was 8.0%, with agreement between the two assays of 98.8%. For endocervical, urethral and male urine samples, agreement was 99.3%, 99.4% and 97.7%, respectively. For ten discrepant samples, alternative amplification assays suggested that the LCx and ProbeTec assays gave erroneous results in six and four cases, respectively. Inhibition of amplification was observed with three (0.25%) urine specimens.

Specific in vivo binding of [125I]-iodomelatonin to melatonin receptors in rat brain.

The in vivo distribution of [125I]-2-iodomelatonin in rat brain was determined at different time intervals after intraarterial injection. After one hour, radioactivity in brain areas devoid of melatonin receptors had washed out to very low concentrations, but significant retention occurred in the medial basal hypothalamus (mbh) which contained the median eminence and in the anterior pituitary gland (ap), areas known to contain high concentrations of melatonin receptors. Coinjection of unlabelled melatonin reduced radioactivity concentrations in the ap and mbh by 44% and 75% respectively at one hour, whereas radiotracer concentrations in other regions remained unchanged. These results indicate the potential for the in vivo study of melatonin receptor concentration changes in human brain in disease states by means of single photon emission computed tomography.

Resolution and in vitro and initial in vivo evaluation of isomers of iodine-125-labeled 1-azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate: a high-affinity ligand for the muscarinic receptor.

1-Azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)- alpha-phenylacetate (IQNP, 1), is a highly selective ligand for the muscarinic acetylcholinergic receptor (mAChR). There are eight stereoisomers in the racemic mixture. The optical isomers of alpha-hydroxy-alpha-phenyl-alpha-(1-propyn-3-yl)acetic acid were resolved as the alpha-methylbenzylamine salts, and the optical isomers of 3-quinuclidinol were resolved as the tartrate salts. The E and Z isomers were prepared by varying the reaction conditions for the stannylation of the triple bond followed by purification utilizing flash column chromatography. In vitro binding assay of the four stereoisomers containing the (R)-(-)-3-quinuclidinyl ester demonstrated that each isomer of 1 bound to mAChR with high affinity. In addition, (E)-(-)-(-)-IQNP demonstrated the highest receptor subtype specificity between the m1 molecular subtype (KD, nM, 0.383 +/- 0.102) and the m2 molecular subtype (29.6 +/- 9.70). In vivo biodistribution studies demonstrated that iodine-125-labeled (E)-(-)-(+)-1 cleared rapidly from the brain and heart. In contrast, iodine-125-labeled (E)-(-)-(-)-, (Z)-(-)-(-)-, and (Z)-(-)-(+)-1 have high uptake and retention in mAChR rich areas of the brain. It was also observed that (E)-(-)-(-)-IQNP demonstrated an apparent subtype selectivity in vivo with retention in M1 (m1, m4) mAChR areas of the rain. In addition, (Z)-(-)-(-)-IQNP also demonstrated significant uptake in tissues containing the M2 (m2) mAChR subtype. These results demonstrate that the iodine-123-labeled analogues of the (E)-(-)-(-)- and (Z)-(-)-(-)-IQNP isomers are attractive candidates for single-photon emission-computed tomographic imaging of cerebral and cardiac mAChR receptor densities.

Compensation for three-dimensional detector response, attenuation and scatter in SPECT grey matter imaging using an iterative reconstruction algorithm which incorporates a high-resolution anatomical image.

The use of SPECT to diagnose physiological alterations in disease states depends on the potential of SPECT to provide a quantitatively accurate reconstructed image. However, the reconstructed values depend upon the shape and size of the brain region as strongly as they depend upon true radioactivity concentration. We report here the results of applying an iterative reconstruction algorithm (IRA) to compensate for shape- and size-dependence, as well as for attenuation and scatter. The IRA is designed only for the reconstruction of images for which the true radioactivity in the white matter within the actual brain is negligible compared with the true radioactivity in the grey matter within the actual brain. The IRA incorporates an accurate three-dimensional model of detector response and utilizes an MRI image which defines the anatomical features of the brain being imaged by segmenting the grey, white and ventricular regions. It is the assumption of radioactivity localization exclusively in the grey matter which permits the efficient incorporation of the MRI image. The IRA was validated by simulation studies that utilized a slice through the basal ganglia in the realistic Hoffman three-dimensional mathematical brain model. FBP images deviate significantly from true radioactivity distribution, whereas IRA images are nearly identical to true radioactivity distribution, except for random fluctuations due to the presence of statistical noise. These results indicate that the application of the IRA will permit SPECT to distinguish deficits due to true physiological changes from apparent deficits due to imaging/reconstruction artifacts.

Three-dimensional simulations of multidetector point-focusing SPECT imaging.

We have applied an efficient algorithm for mathematically simulating the three-dimensional (3-D) response of a SPECT imaging system with a depth-dependent 3-D point spread function (3-DPSF). The input object whose reconstructed image is to be simulated is restricted to a binary map; more complex objects may be treated as linear combinations of binary maps. The 3-D convolution reduces to a sequence of additions of a 3-D line spread function (3-DLSF), appropriately translated, to the 3-D response. We have simulated the projection data from a multidetector SPECT system with point-focusing collimators. The simulated projection data were then reconstructed using the manufacturer's software. The objects simulated included simple geometrical solids such as spheres and cylinders, as well as the distribution of muscarinic cholinergic receptors in a realistic brain slice. The results of these simulations indicate the existence of significant qualitative and quantitative artifacts in reconstructed human brain images.

Three-dimensional SPECT simulations of a complex three-dimensional mathematical brain model and measurements of the three-dimensional physical brain phantom.

We have developed a three-dimensional computer simulation of SPECT imaging. We have applied the simulation procedure to the realistic mathematical Hoffman three-dimensional brain model to generate the projection data (in the absence of attenuation, scatter, or noise) of both a parallel-hole and a multidetector SPECT system with point-focusing collimators. The simulated projection data were then reconstructed using standard software. The projection data resulting from the distribution of grey matter alone, or grey and white matter, were simulated. The results of these simulations indicate the existence of significant qualitative and quantitative artifacts in reconstructed human brain images. For example, the reconstructed values for grey matter along a cortical circumferential profile in a transverse slice through the basal ganglia varied by a factor of 2.40 (parallel-hole) and 2.99 (point-focusing), although the original grey matter values were identical in all cortical regions in the model. We have compared the simulated reconstructed images with those obtained by imaging the physical three-dimensional Hoffman brain phantom, which was constructed based upon the same set of data from which the mathematical three-dimensional Hoffman brain model was derived. Although the simulation did not include all of the degrading factors present in the physical imaging, the two images were in good agreement, indicating the applicability of the simulation to a realistic situation and the importance of the detector resolution effect.

In vivo competition studies of Z-(-,-)-[125I]IQNP against 3-quinuclidinyl 2-(5-bromothienyl)-2-thienylglycolate (BrQNT) demonstrating in vivo m2 muscarinic subtype selectivity for BrQNT.

Alzheimer's disease (AD) involves selective loss of muscarinic m2, but not m1, subtype neuroreceptors in cortical and hippocampal regions of the human brain. Until recently, emission tomographic study of the loss of m2 receptors in AD has been limited by the absence of available m2-selective radioligands that can penetrate the blood-brain barrier. We now demonstrate the in vivo m2 selectivity of an analog of (R)-QNB, 3-quinuclidinyl 2-(5-bromothienyl)-2-thienylglycolate (BrQNT), by dissection and autoradiographic studies of the in vivo inhibition of radioiodinated Z-1-azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenyl-acetate (Z-(-,-)-[125I]IQNP) binding by unlabeled BrQNT in rat brain. In the absence of BrQNT, Z-(-,-)-[125I]IQNP labels brain regions containing muscarinic receptors, with an enhanced selectivity for the m2 subtype. In the presence of 60-180 nmol of co-injected racemic BrQNT, Z-(-,-)-[125I]IQNP labeling in those brain regions containing predominantly m2 subtype is reduced to background levels, while levels of radioactivity in areas not enriched in the m2 subtype do not significantly decrease. We conclude that BrQNT is m2-selective in vivo, and that [76Br]BrQNT, or a radiofluorinated analog, may be of potential use in positron emission tomographic (PET) study of the loss of m2 receptors in AD. In addition, a radioiodinated analog may be of potential use in single photon emission tomographic (SPECT) studies.

In vivo dissociation kinetics of [3H]quinuclidinyl benzilate: relationship to muscarinic receptor concentration and in vitro kinetics.

The in vivo washout kinetics of [3H]quinuclidinyl benzilate ([3H]QNB) varies significantly in various structures in the rat brain. The slowest washout rates are from the hippocampus, corpus striatum, and cortex, intermediate rates are exhibited from the thalamus and colliculi, while the fastest washout rate is from the cerebellum. We have also demonstrated a difference in the in vitro dissociation rates (k-1) of [3H]QNB from various structures. The k-1 for the hippocampus, corpus striatum and cortex, is two-fold slower than that observed in the thalamus, colliculi, and cerebellum. The differences in the in vitro dissociation kinetics are not, however, sufficient to explain the differences in the in vivo washout kinetics. We have developed a theoretical formulation which describes conditions under which the washout kinetics are a function of the concentration of receptor in a structure. Furthermore, we present a graphical method in which a plot of the reciprocal of the observed washout rate constant, 1/k(obs), vs receptor concentration is linear. Analysis of the washout kinetics of [3H]QNB from various structures of the CNS of rat were well described by this theory when the differences in in vitro k-1 are included.

In vitro and in vivo m2 muscarinic subtype selectivity of some dibenzodiazepinones and pyridobenzodiazepinones.

Alzheimer's disease (AD) involves selective loss of muscarinic m2, but not m1, subtype receptors in cortical and hippocampal regions of the human brain. Emission tomographic study of the loss of m2 receptors in AD has been limited by the absence of available m2-selective radioligands, which can penetrate the blood-brain barrier. We now report on the in vitro and in vivo m2 muscarinic subtype selectivity of a series of dibenzodiazepinones and pyridobenzodiazepinones determined by competition studies against (R)-3-quinuclidinyl (S)-4-iodobenzilate ((R,S)-[125I]IQNB) or [3H]QNB. Of the compounds examined, three of the 5-[[4-[(4-dialkylamino)butyl]-1-piperidinyl]acetyl]-10, 11-dihydro-5-H-dibenzo[b,e][1,4]diazepin-11-ones (including DIBA) and three of the 11-[[4-[4-(dialkylamino)butyl]-1-phenyl]acetyl]-5, 11-dihydro-6H-pyrido [2,3-b][1,4]benzodiazepin-6-ones (including PBID) exhibited both high binding affinity for the m2 subtype (/=10). In vivo rat brain dissection studies of the competition of PBID or DIBD against (R,S)[125I]IQNB or [3H]QNB exhibited a dose-dependent preferential decrease in the binding of the radiotracer in brain regions that are enriched in the m2 muscarinic subtype. In vivo rat brain autoradiographic studies of the competition of PBID, BIBN 99, or DIBD against (R,S)[125I]IQNB exhibited an insignificant effect of BIBN 99 and confirmed the effect of PBID and DIBD in decreasing the binding of (R,S)[125I]IQNB in brain regions that are enriched in the m2 muscarinic subtype. We conclude that PBID and DIBD are potentially useful parent compounds from which in vivo m2 selective derivatives may be prepared for potential use in positron emission tomographic (PET) study of the loss of m2 receptors in AD.

Characterization of in vivo brain muscarinic acetylcholine receptor subtype selectivity by competition studies against (R,S)-[125I]IQNB.

We have studied the in vivo rat brain muscarinic acetylcholine receptor (mAChR) m2 subtype selectivities of three quinuclidine derivatives: (R)-3-quinuclidinyl benzilate (QNB), E-(+,+)-1-azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (E-(+,+)-IQNP), and E-(+,-)-1-azabicyclo[2.2.2]oct-3-yl alpha-hydroxy-alpha-(1-iodo-1-propen-3-yl)-alpha-phenylacetate (E-(+,-)-IQNP), and two tricyclic ring compounds: 5-[[4-[4-(diisobutylamino)butyl]-1-phenyl]-10,11-dihydro-5H-dibenz o [b,e][1,4]diazepin-11-one [sequence: see text] (DIBD) and 11-[[4-[4-(diisobutylamino)butyl-1-phenyl]acetyl]-5,11-dihydro-6H- pyrido [2,3-b][1,4]benzodiazepin-6-one [sequence: see text] (PBID), by correlating the regional inhibition of (R,S)-[125I]IQNB with the regional composition of the m1-m4 subtypes. Subtle effects are demonstrated after reduction of the between-animal variability by normalization to corpus striatum. Substantial in vivo m2-selectivity is exhibited by QNB and DIBD, modest in vivo m2-selectivity is exhibited by E-(+,+)-IQNP, and little or no in vivo m2-selectivity is exhibited by PBID and E-(+,-)-IQNP. Surprisingly, the in vivo m2-selectivity is not correlated with the in vitro m2-selectivity. For example, QNB, which appears to be the most strongly in vivo m2-selective compound, exhibits negligible in vitro m2-selectivity. These examples indicate that a strategy which includes only preliminary in vitro screening may very well preclude the discovery of a novel compound which would prove useful in vivo.

Pharmacokinetic computer simulations of the relationship between in vivo and in vitro neuroreceptor subtype selectivity of radioligands.

Pharmacokinetic computer simulations reveal a discrepancy between the in vivo and in vitro neuroreceptor subtype selectivity of radioligands. For radioligands with an in vitro neuroreceptor subtype selectivity between 0.1 and 10.0, the in vivo neuroreceptor subtype selectivity appears to be constrained to be between 0.1 and 10.0, but, in general, is not equal to the in vitro selectivity. For example, if the in vitro selectivity is 1.0 (that is, the radioligand is nonselective in vitro) the in vivo selectivity may be thought of as a random variable having a significant nonzero probability for values as low as 0.1 or as high as 10.0, with a moderate peak at a value of 1.0. For a radioligand whose in vitro subtype selectivity is greater than 10.0, the in vivo selectivity is bounded above by the in vitro subtype selectivity, but may be several orders of magnitude lower than the in vitro subtype selectivity. Thus, in spite of the discrepancy between the in vivo and in vitro neuroreceptor subtype selectivity of radioligands, there are two useful inferences about the in vivo selectivity that might be drawn from knowledge of the in vitro selectivity: (1) If the in vitro selectivity is between 0.1 and 10.0, then, at best, the in vivo selectivity might be as high as 10.0. (2) If the in vitro selectivity is greater than 10.0, then, at best, the in vivo selectivity might be as high as the in vitro selectivity.

Autoradiographic evidence that (R)-3-quinuclidinyl (S)-4-fluoromethylbenzilate ((R,S)-FMeQNB) displays in vivo selectivity for the muscarinic m2 subtype.

Alzheimer's disease (AD) involves selective loss of muscarinic m2, but not m1, subtype neuroreceptors in cortical and hippocampal regions of the human brain. Until recently, emission tomographic study of the loss of m2 receptors in AD has been limited by the absence of available m2-selective radioligands that can penetrate the blood-brain barrier. We now demonstrate the in vivo m2 selectivity of a fluorinated derivative of QNB, (R)-3-quinuclidinyl (S)-4-fluoromethylbenzilate ((R,S)-FMeQNB), by studying autoradiographically the in vivo inhibition of radioiodinated (R)-3-quinuclidinyl (S)-4-iodobenzilate ((R,S)-[125I]IQNB) binding by unlabelled (R,S)-FMeQNB. In the absence of (R,S)-FMeQNB, (R,S)-[125I]IQNB labels brain regions in proportion to the total muscarinic receptor concentration; in the presence of 75 nmol of (R,S)-FMeQNB, (R,S)-[125I]IQNB labelling in those brain regions containing predominantly m2 subtype is reduced to background levels. We conclude that (R,S)-FMeQNB is m2-selective in vivo, and that (R,S)-[18F]FMeQNB may be of potential use in positron emission tomographic (PET) study of the loss of m2 receptors in AD.

Specific binding component of the "inactive" stereoisomer (S,S)-[125I] IQNB to rat brain muscarinic receptors in vivo.

In vivo nonspecific binding can be estimated using the inactive stereoisomer of a receptor radioligand. However, the binding of the inactive stereoisomer may be partially specific. Specific binding of the inactive (S,S)-[125I]IQNB was estimated from the inhibition induced by a competing nonradioactive ligand. This technique differed from the usual approach, since it was used to study the inactive rather than the active stereoisomer. The results indicate that there is substantial specific binding for (S,S)-[125I]IQNB.

Evaluation of 1-azabicyclo[2.2.2]oct-3-yl alpha-fluoroalkyl-alpha-hydroxy-alpha-phenylacetates as potential ligands for the study of muscarinic receptor density by positron emission tomography.

Both 1-azabicyclo[2.2.2]oct-3-yl alpha-(1-fluoroeth-2-yl)-alpha-hydroxy-alpha-phenylacetate (FQNE, 5) and 1-azabicyclo[2.2.2]oct-3-yl alpha-(1-fluoropent-5-yl)-alpha-hydroxy-alpha-phenylacetate (FQNPe, 6) were prepared and evaluated as potential candidates for the determination of muscarinic cholinergic receptor (mAChR) density by positron emission tomography (PET). The results of in vitro binding assays demonstrated that although both 5 and 6 had high binding affinities for m1 and m2 mAChR subtypes, 6 displayed a higher affinity (nM, m1; KD, 0.45, m2; KD, 3.53) as compared to 5 (nM, m1; KD, 12.5, m2; KD, 62.8). It was observed that pretreatment of female Fisher rats with either 5 or 6 prior to the i.v. administration of Z-(-)(-)-[131I]-IQNP, a high-affinity muscarinic ligand, significantly blocked the uptake of radioactivity in the brain and heart measured 3 h postinjection of the radiolabeled ligand. These new fluoro QNB analogues represent important target ligands for evaluation as potential receptor imaging agents in conjunction with PET.

Pharmacokinetic simulations of SPECT quantitation of the M2 muscarinic neuroreceptor subtype in disease states using radioiodinated (R,R)-4IQNB.

Alzheimer's disease (AD) involves selective loss of muscarinic M2, but not M1, subtype neuroreceptors in the posterior parietal cortex of the human brain. Emission tomographic study of the loss of M2 receptors in AD is limited by the fact that there is currently no available M2-selective radioligand which can penetrate the blood-brain barrier. However, by taking advantage of the different pharmacokinetic properties of (R,R)-[123I]IQNB for the M1 and M2 subtypes, it may be possible to estimate losses in M2. It has previously been hypothesized that the difference between an early study and a late study should provide information on the M2 receptor population. In order to test this hypothesis, we present here the results of pharmacokinetic simulations of the in vivo localization of (R,R)-[123I]IQNB in brain regions containing various proportions of M1 and M2 subtypes. These results permit us to conclude that SPECT imaging of (R,R)-[123I]IQNB localization can potentially be used to quantitate changes in the M2 subtype in a disease state within a brain region for which the ratio M2/M1 is sufficiently high in normal individuals.

Muscarinic receptor selectivities of 3-Quinuclidinyl 8-xanthenecarboxylate (QNX) in rat brain.

We have determined the binding of (R)-3-Quinuclidinyl 8-xanthenecarboxylate to muscarinic acetylcholine receptor preparations from rat cortex, hippocampus, caudate/putamen, thalamus, pons and colliculate bodies. The competition curves determined with [3H]quinuclidinyl benzilate as the radioligand are well described by a two site model with a difference in affinity between the two sites of 12-fold. The proportions of high affinity site vary from 100% in the caudate/putamen to 0% in the pons/medulla. The selectivities are different from those measured by pirenzepine and are consistent with QNX exhibiting similar affinity for the M1, M3, and M4 receptors with lower affinity for the M2 receptor. This assignment was confirmed by determining the affinities of QNX for the cloned receptor subtypes.

[3H]QNB displays in vivo selectivity for the m2 subtype.

Alzheimer's disease (AD) involves selective loss of muscarinic m2, but not m1, subtype neuroreceptors in the posterior parietal cortex of the human brain. Emission tomographic study of the loss of m2 receptors in AD is limited by the fact that there is currently no available m2-selective radioligand which can penetrate the blood-brain barrier. [3H](R)-3-quinuclidinylbenzilate ([3H]QNB) is commonly used for performing in vitro studies of the muscarinic acetylcholine receptor (mAChR), either with membrane homogenates or with autoradiographic slices, in which [3H]QNB is nonsubtype-selective. We report here the results of in vivo studies, using both carrier-free and low specific activity [3H]QNB, which show that [3H]QNB exhibits a substantial in vivo m2-selectivity. Previously reported in vivo (R)-3-quinuclidinyl (R)-4-iodobenzilate ((R,R)-[125I]IQNB) binding appears to be nonsubtype-selective. Apparently the bulky iodine substitution in the 4 position reduces the subtype selectivity of QNB. It is possible that a less bulky fluorine substitution might permit retention of the selectivity exhibited by QNB itself. We conclude that a suitably radiolabeled derivative of QNB, possibly labeled with 18F, may be of potential use in positron emission tomographic (PET) study of the loss of m2 receptors in AD.

A novel muscarinic receptor ligand which penetrates the blood brain barrier and displays in vivo selectivity for the m2 subtype.

Alzheimer's disease (AD) involves selective loss of muscarinic m2, but not m1, subtype neuroreceptors in the posterior parietal cortex of the human brain. Emission tomographic study of the loss of m2 receptors in AD is limited by the fact that there is currently no available m2-selective radioligand which can penetrate the blood-brain barrier. In our efforts to prepare such a radioligand, we have used competition studies against currently existing muscarinic receptor radioligands to infer the in vitro and in vivo properties of a novel muscarinic receptor ligand, 5-[[4-[4-(diisobutylamino)butyl]-1-phenyl]acetyl]-10,11-dihydro-5H - -dibenzo [b,e][1,4]diazepin-11-one (DIBD). In vitro competition studies against [3H](R)-3-quinuclidinylbenzilate ([3H]QNB) and [3H]N-methylscopolamine ([3H]NMS), using membranes derived from transfected cells expressing only m1, m2, m3, or m4 receptor subtypes, indicate that DIBD is selective for m2/m4 over m1/m3. In vivo competition studies against (R,R)-[125I]IQNB indicate that DIBD crosses the blood brain barrier (BBB). The relationship of the regional percentage decrease in (R,R)-[125I]IQNB versus the percentage of each of the receptor subtypes indicates that DIBD competes more effectively in those brain regions which are known to be enriched in the m2, relative to the m1, m3, and m4, receptor subtype; however, analysis of the data using a mathematical model shows that caution is required when interpreting the in vivo results. We conclude that a suitably radiolabeled derivative of DIBD may be of potential use in emission tomographic study of changes in m2 receptors in the central nervous system.

Theoretical relationships of receptor and delivery sensitivities and measurable parameters in in vivo neuroreceptor-radioligand interactions.

In vivo quantification of neuroreceptors in human brains by PET or SPECT is complicated by the fact that a number of variables other than receptor concentration may influence the observed radioactivity in a brain region. This consideration has led the authors to formulate rigorous mathematical definitions of the concepts of receptor and delivery sensitivities. It has been speculated that a neuroreceptor-radioligand system having a high (low) receptor sensitivity would have a low (high) delivery sensitivity, and that the receptor sensitivity of a neuroreceptor-radioligand system can be determined by observing the time-course of the brain radioligand concentration following injection of no carrier added (nca) radioligand. Computer simulation studies of the characteristics of a simple model for in vivo neuroreceptor-radioligand interaction show that, under a set of realistic restrictions, there is a unique and intuitively satisfying relationship between receptor and delivery sensitivities: receptor sensitivity+delivery sensitivity approximately 1. In addition, the receptor sensitivity can be computed as a function of the observable parameters of the nca radioligand time course. These straightforward relationships are surprising in light of the complexity of the analytical solutions.