Equivalent dynamic human brain NK1-receptor occupancy following single-dose i.v. fosaprepitant vs. oral aprepitant as assessed by PET imaging.

The type 1 neurokinin receptor (NK1R) antagonist aprepitant and its i.v. prodrug fosaprepitant have been approved for prevention of acute and delayed nausea and vomiting associated with chemotherapy. This study evaluated the magnitude and duration of brain NK1R occupancy over a period of 5 days after single-dose i.v. infusion of 150-mg fosaprepitant and single-dose oral administration of 165-mg aprepitant, using serial [(18)F]MK-0999 positron emission tomography (PET) in 16 healthy subjects. Each subject underwent three scans. Brain NK1R occupancy rates after i.v. fosaprepitant at time to peak concentration (T(max); ~30 min), 24, 48, and 120 h after the dose were 100, 100, ≥97, and 41-75%, respectively. After aprepitant, NK1R occupancy rates at these time points (T(max) ~4 h) were ≥99, ≥99, ≥97, and 37-76%, respectively. Aprepitant plasma concentration profiles were comparable for the two dosage forms. The study illustrates the utility of PET imaging in determining central bioequivalence in a limited number of subjects.

Nonpeptide alpha(v)beta(3) antagonists. 1. Transformation of a potent, integrin-selective alpha(IIb)beta(3) antagonist into a potent alpha(v)beta(3) antagonist.

Modification of the potent fibrinogen receptor (alpha(IIb)beta(3)) antagonist 1 generated compounds with high affinity for the vitronectin receptor alpha(v)beta(3). Sequential modification of the basic N-terminus of 1 led to the identification of the 5,6,7, 8-tetrahydro[1,8]naphthyridine moiety (THN) as a lipophilic, moderately basic N-terminus that provides molecules with excellent potency and selectivity for the integrin receptor alpha(v)beta(3). The THN-containing analogue 5 is a potent inhibitor of bone resorption in vitro and in vivo. In addition, the identification of a novel, nonpeptide radioligand with high affinity to alpha(v)beta(3) is also reported.

Intraoperative parathyroid aspiration and parathyroid hormone assay as an alternative to frozen section for tissue identification.

Most people would agree that successful parathyroidectomy depends on two important variables: the surgeon's recognition and excision of the abnormal parathyroid gland(s) and the pathologist's confirmation that the removed tissue is parathyroid tissue. Frozen section is usually employed to confirm the identity of parathyroid tissue, but occasionally confirmation cannot be made without a permanent section, as with intrathyroidal glands. This study proposes a new method of expeditious and easy confirmation of parathyroid tissue utilizing the immunoassay for quick measurement of intraoperative parathyroid hormone (IOPTH). By directly aspirating the suspected adenoma, the assay becomes a rapid diagnostic tool that can be used as an alternative to frozen section. In cases where the surgeon is already planning to employ the assay, the elimination of frozen section is cost-effective. Intraoperative aspiration of histologically confirmed parathyroid adenomas was performed on 12 consecutive patients undergoing parathyroid surgery. Parathyroid glands were aspirated with a 22-gauge syringe after gland excision. Aspirates were placed in 1 to 3 ml of buffered saline. A similar process was performed on 12 thyroid controls. Specimens were centrifuged, aliquotted, and stored at -70 degrees C. The parathyroid hormone value was analyzed electively by rapid assay and the values recorded. For all parathyroid aspirates, the rapid assay value was > 1500 pg/ml, exceeding the uppermost limit of the diagnostic chart. Values for thyroid aspirates ranged from 58 to 85 pg/ml (mean 75.7 pg/ml). In all cases tissue confirmation was achieved with permanent section. Values were 100% sensitive and specific. Measurement of PTH from intraoperative aspiration of suspected parathyroid adenomas is clinically useful in patients for whom frozen section would routinely be employed. Values > 1500 pg/ml secure the tissue diagnosis. There is no additional cost in cases where IOPTH monitoring is already being utilized to confirm cure. The elimination of frozen section could be cost-effective and, for some institutions, actually decrease the operating time as the IOPTH assay takes only 15 minutes. PTH assay is an accurate diagnostic technique and to date is 100% sensitive and specific for differentiating between parathyroid tumors and thyroid nodules.

HIV-1 integrase inhibitors that compete with the target DNA substrate define a unique strand transfer conformation for integrase.

Diketo acids such as L-731,988 are potent inhibitors of HIV-1 integrase that inhibit integration and viral replication in cells. These compounds exhibit the unique ability to inhibit the strand transfer activity of integrase in the absence of an effect on 3' end processing. To understand the reasons for this distinct inhibitory profile, we developed a scintillation proximity assay that permits analysis of radiolabeled inhibitor binding and integrase function. High-affinity binding of L-731,988 is shown to require the assembly of a specific complex on the HIV-1 long terminal repeat. The interaction of L-731,988 with the complex and the efficacy of L-731, 988 in strand transfer can be abrogated by the interaction with target substrates, suggesting competition between the inhibitor and the target DNA. The L-731,988 binding site and that of the target substrate are thus distinct from that of the donor substrate and are defined by a conformation of integrase that is only adopted after assembly with the viral end. These results elucidate the basis for diketo acid inhibition of strand transfer and have implications for integrase-directed HIV-1 drug discovery efforts.

Non-invasive radiotracer imaging as a tool for drug development.

Non-Invasive Radiotracer Imaging (NIRI) uses either short-lived positron-emitting isotopes, such as 11C and 18F, for Positron Emis ion Tomography (PET) or single photon emitting nuclides, e.g., 123I, which provide images using planar imaging or Single-Photon Emission Computed Tomography (SPECT). These high-resolution imaging modalities provide anatomical distribution and localization of radiolabeled drugs, which can be used to generate real time receptor occupancy and off-rate studies in humans. This can be accomplished by either isotopically labeling a potential new drug (usually with 11C), or indirectly by studying how the unlabelled drug inhibits specific radioligand binding in vivo. Competitive blockade studies can be accomplished using a radiolabeled analogue which binds to the site of interest, rather than a radiolabeled version of the potential drug. Imaging, particularly PET imaging, can be used to demonstrate the effect of a drug through a biochemical marker of processes such as glucose metabolism or blood flow. NIRI as a development tool in the pharmaceutical industry is gaining increased acceptance as its unique ability to provide such critical information in human subjects is recognized. This section will review recent examples that illustrate the utility of NIRI, principally PET, in drug development, and the potential of imaging advances in the development of cancer drugs and gene therapy. Finally, we provide a brief overview of the design of new radiotracers for novel targets.

Positron emission tomography neuroreceptor imaging as a tool in drug discovery, research and development.

Improved communication and cooperation between research-driven drug companies and academic positron emission tomography (PET) centers, coupled with improvements in PET camera resolution, the availability of small animal PET cameras and a growing list of neuroreceptor-specific PET tracers, have all contributed to a substantial increase in the use and value of PET as a tool in central nervous system drug discovery and development.

Investigation of angiotensin II/AT1 receptors with carbon-11-L-159,884: a selective AT1 antagonist.

UNLABELLED: Antagonists of the angiotensin II AT1 receptor subtype have been recently introduced for treatment of arterial hypertension and for pharmacological studies of these receptors. The purpose of this work was to label such an antagonist with 11C and test the applicability of the radioligand for PET studies. METHODS: The potent and selective nonpeptide AT1 antagonist L-159,884 was labeled with 11C and injected intravenously into six dogs. Renal accumulation and kinetics of the radioligand were imaged with PET at baseline and after receptor blockade with 1 mg/kg MK-996. Time-activity curves were derived from the renal cortex and were analyzed by the Gjedde-Patlak plot to obtain the influx rate constant of the radioligand. RESULTS: There was selective radioligand binding in the kidneys, mainly located in the cortex. Within the time interval between 95 and 115 min postinjection, the radioactivity retained in the kidneys was 109 +/- 27 and 42 +/- 4 nCi/ml/mCi of the injected dose for the control and inhibition studies, respectively. The influx rate constant of the radioligand decreased from a baseline of 0.0298 +/- 0.0156 to a post-MK-996 value of 0.0098 +/- 0.0052. CONCLUSION: These results demonstrate distinct binding of 11C-L-159,884 in the renal cortex with a specific binding component suitable for quantitative PET imaging of angiotensin II/AT1 receptors.

Identification of low molecular weight GP IIb/IIIa antagonists that bind preferentially to activated platelets.

A critical function of fibrinogen in hemostasis and thrombosis is to mediate platelet aggregation by binding selectively to an activated form of glycoprotein (GP) IIb/IIIa. Although numerous peptide and nonpeptide fibrinogen receptor antagonists have been described, their binding selectivity for resting and activated platelets has not been explored. Therefore, dissociation constants of GP IIb/IIIa antagonists for two biochemically separated forms of purified GP IIb/IIIa and for resting and activated platelets were determined by competitive displacement of the dansyl fluorophore containing GP IIb/IIIa antagonist L-736,622. Also, coating either form of the purified GP IIb/IIIa onto yttrium silicate scintillation proximity assay fluomicrospheres produced an activated form of the receptor, whose binding affinity for GP IIb/IIIa antagonists was measured conveniently by competition with the arginine-glycine-aspartic acid (RGD) containing heptapeptide [125I]L-692,884. In addition, direct binding measurements with radiolabeled GP IIb/IIIa antagonists also were performed on resting and activated platelets. We identified two classes of compounds. One class binds to both forms of GP IIb/IIIa, as well as resting and activated platelets, with similar Kd values (e.g., L-736,622 and Echistatin). The other class of compounds binds with much higher affinity to the activated form of GP IIb/IIIa (purified or on platelets) as compared with the resting form (e.g., L-734,217, MK-852, tirofiban and L-692,884). Selective antagonists, like L-734,217 (KdActivated = 5 nM and KdResting = 620 nM), can effectively inhibit ex vivo platelet aggregation at concentrations of drug that produce low levels of occupancy of the circulating platelet receptors. The potential clinical advantages of selective versus nonselective GP IIb/IIIa antagonists remain to be explored in clinical trials.

Non-peptide glycoprotein IIb/IIIa inhibitors. 17. Design and synthesis of orally active, long-acting non-peptide fibrinogen receptor antagonists.

The synthesis and pharmacological evaluation of 5 (L-738, 167), a potent, selective non-peptide fibrinogen receptor antagonist is reported. Compound 5 inhibited the aggregation of human gel-filtered platelets with an IC50 value of 8 nM and was found to be > 33000-fold less effective at inhibiting the attachment of human endothelial cells to fibrinogen, fibronectin, and vitronectin than it was at inhibiting platelet aggregation. Ex vivo platelet aggregation was inhibited by > 85% 24 h after the oral administration of 5 to dogs at 100 micrograms/kg. The extended pharmacodynamic profile exhibited by 5 appears to be a consequence of its high-affinity binding to GPIIb/IIIa on circulating platelets and suggests that 5 is suitable for once-a-day dosing.

Kinetics of the conjugation of aniline mustards with glutathione and thiosulfate.

The rates of the non-enzymatic conjugation of the substituted aniline mustards, melphalan, chlorambucil and p-(N,N-bis(2-chloroethyl))toluidine with glutathione and thiosulfate were determined using nuclear magnetic resonance spectroscopy. Using this method, the disappearance of drug and the formation of both the mono-thioether and bis-thioether conjugates can be monitored directly. For glutathione conjugation, the rate constants for the formation of the first and second aziridinium intermediates were similar. With thiosulfate conjugation, the rate constant for the formation of the first aziridinium intermediate is greater than the rate constant for the formation of the second aziridinium. This demonstrates that the type of nucleophile has a significant influence on the overall alkylating activity of these bifunctional mustards. The bisthioether adduct formed from the reaction between p-(N,N-bis([2-13C]-2-chloroethyl))toluidine and glutathione and thiosulfate can be identified and scrambling of the 13C label in the product provides strong evidence that the alkylation must occur through an aziridinium intermediate.

Disposition of L-738,167, a potent and long-acting fibrinogen receptor antagonist, in dogs. Dose-dependent pharmacokinetics.

L-738,167 is a potent and long-acting fibrinogen receptor antagonist and may be useful for treatment of chronic thrombotic occlusive disorders. The purposes of this study were to characterize the metabolism and disposition of L-738,167, and to investigate factors affecting its pharmacokinetic behaviors in dogs, one of the animal models used in pharmacological and toxicological studies. In vitro and in vivo experiments indicated that L-738,167 was not metabolized to any appreciable extent in dogs. Biliary excretion was found to be the major route (approximately 75%) of drug elimination. Following 1 and 3 micrograms/kg iv doses, blood pharmacokinetics of L-738,167 were linear. Total blood clearance (CLB) was much lower than hepatic blood flow, and the apparent volume of distribution at steady-state (Vdss,B) was comparable with blood volume. Blood pharmacokinetics in the dose range of 3-250 micrograms/kg were dose-dependent; both CLB and Vdss,B for L-738,167 increased markedly with increasing doses. However, the terminal half-life (t1/2) was dose-independent, with a mean value of approximately 4 days. L-738,167 was found to bind negligibly to dog plasma proteins. Determinations of whole blood (WB), platelet-rich plasma, and platelet-poor plasma concentrations after several intravenous doses of [3H]L-738,167 revealed significant concentration-dependent binding of the compound to platelets. Kinetic analysis of the platelet binding indicated that L-738,167 was bound to dog platelets with high affinity (apparent Kd approximately 1 nM platelet-poor plasma concentration) and relatively low capacity (approximately 70 nM WB concentration). Findings are consistent with the binding kinetics of L-738,167 to glycoprotein IIb/IIIa (GP IIb/IIIa) receptor, supporting that GP IIb/IIIa was the primary binding component on the platelets. It was concluded that the dose-dependent pharmacokinetics of L-738,167 were the consequence of the concentration-dependent drug-platelet binding. Due to this extensive platelet binding, L-738,167, when given in therapeutic doses or lower, resided primarily in the vascular compartment-the site of pharmacological action. At doses exceeding the receptor binding capacity, the excess amount or the unbound drug was eliminated rapidly. In all cases, the equally long t1/2 of L-738,167 was also a consequence of the high-affinity binding to platelets, in good agreement with its prolonged pharmacodynamic profile.

A case of composite Hodgkin's disease and chronic lymphocytic leukemia in bone marrow. Lack of Epstein-Barr virus.

We report Hodgkin's disease arising in a 68-year-old patient with a history of chronic lymphocytic leukemia for 8 years. The patient presented with a 4-month history of weakness, loss of appetite, and a 15-pound weight loss. A bone marrow biopsy showed two distinct histologic types of lymphoma: chronic lymphocytic leukemia and Hodgkin's disease. Immunohistochemical studies showed that chronic lymphocytic leukemia cells were composed of kappa-light chain-restricted monoclonal B cells. The Reed-Sternberg cells expressed CD15. Epstein-Barr virus RNA was not identified in either the Reed-Sternberg cells or cells of chronic lymphocytic leukemia by in situ hybridization. To our knowledge, this is the second reported case of composite Hodgkin's disease and chronic lymphocytic leukemia involving the bone marrow.

Development of [11C]L-159,884: a radiolabelled, nonpeptide angiotensin II antagonist that is useful for angiotensin II, AT1 receptor imaging.

[11C]L-159,884 ([11C] N-[[4'[(2-ethyl-5,7-dimethyl-3H- imidazo[4,5-b]pyridin-3-yl) methyl] [1,1'-biphenyl]-2-yl] sulfonyl]-4-methoxybenzamide) and [11C]L-162,574 ([11C] N-[[4'[2-ethyl-5,7- dimethyl-3H-imidazo[4,5-b] pyridin-3-yl)methyl] [1,1'-biphenyl]-2-yl]sulfonyl]-3- methoxybenzamide), both potent and selective ligands for the AT1 receptor, were prepared by C-11 methylation of the corresponding desmethyl phenolic precursors. The radiotracers were purified by semi-preparative reverse-phase HPLC. Non-decay corrected radiochemical yields were 5 and 3% for L-159,884 and L-162,574 respectively, and the average specific activity was 2979 mCi/mumol at end-of-synthesis (EOS). The average time of synthesis was 18 min.

In vivo labeling of angiotensin II receptors with a carbon-11-labeled selective nonpeptide antagonist.

UNLABELLED: Angiotensin II (ANG II) initiates a variety of physiological effects by binding to high affinity receptors. Two ANG II receptor subtypes, AT1 and AT2, have recently been identified. This study was undertaken to evaluate [11C]L-159,884, an AT1 subtype selective nonpeptide antagonist, as a potential PET tracer. METHODS: Carbon-11-L-159,884 was prepared by alkylation of the nor precursor with [11C]methyliodide and was studied for its in vivo binding characteristics, biodistribution and kinetics in mice. The effects of PD-123319, an AT2-selective ANGII antagonist, as well as those of alpha- and beta-adrenergic drugs on [11C]L-159,884 binding were investigated also. RESULTS: Administration of the AT1 antagonists resulted in dose-dependent inhibition of [11C]L-159,884 binding in the kidneys, the organ with the highest density of AT1 receptors. Inhibition was also observed in the lungs and the heart. Adrenergic drugs did not influence [11C]L-159,884 binding to AT1 receptors. Kinetic studies showed rapid tracer uptake in the liver, kidneys, lungs and heart. Excretion of the radioactivity occurred primarily through the intestinal tract (> 20% in 90 min), with less than 8% excreted through the urine. CONCLUSION: The results suggest that [11C]L-159,884 binds in vivo to AT1 receptors in mouse kidneys, lungs and heart. This radiotracer appears to be a promising candidate for studying ANG II receptors in vivo by PET.

Blood specimens from patients referred for cytogenetic analysis: Vanderbilt University experience from 1985 to 1992.

Cytogenetic records were examined from consecutive nononcology blood specimens from 2,821 patients referred for cytogenetic services to Vanderbilt University Medical Center, Nashville, Tenn, from January 1985 to December 1992. We grouped the records according to reasons for referral and diagnoses. The most common reasons for referral were history of multiple abortions/miscarriages (23.3%), possibility of chromosomal abnormality (18.8%), and possible presence of the fragile X syndrome (15.6%). Overall, 2,418 (85.7%) patients were found to have normal chromosomes, and 403 (14.3%) patients were diagnosed with a cytogenetic abnormality. For example, 20 (5.4%) of the 373 males referred for the fragile X syndrome, or 1.4% of all males (20 of 1,428) excluding those with ambiguous genitalia, were diagnosed with this syndrome while 8 (2.1%) of the 373 males had a chromosome abnormality other than the fragile X chromosome. In addition, 85 (70.2%) of 121 males referred for Down syndrome had this syndrome, and only 53 (40.8%) of 130 females referred for Down syndrome had this diagnosis. This study should assist physicians in middle Tennessee and surrounding areas by increasing their awareness of the types and frequencies of cytogenetic diseases and by providing figures for comparison with other regions of the country.

Differences in localization ability in cases of right versus left unilateral simulated conductive hearing loss.

The purpose of this study was to determine whether localization errors were greater in cases of right ear unilateral simulated conductive loss, as compared to left ear simulated conductive loss of the same degree and configuration. Thirty adults were asked to localize a 3-kHz warbled pure tone with their left and right ears alternately occluded with an earplug. Significantly greater localization difficulty was found when the right ear was occluded. The average error was 58 degrees with the left ear plugged and 64 degrees when the right ear was occluded, which approximates the 65-degree average error that would have resulted from random guessing. This suggests that short-term conductive losses result in unusually poor localization ability, which is worse if the right ear is affected.

Comparison of binding to rapidly activating delayed rectifier K+ channel, IKr, and effects on myocardial refractoriness for class III antiarrhythmic agents.

Saturation binding studies in guinea pig ventricular myocytes with 3H-dofetilide, a radioligand for the cardiac rapidly activating delayed rectifier K+ IKr channel, indicated specific high-affinity binding with a Kd of 83 nM and a Bmax of 0.18 pmol/mg cellular protein (1.36 x 10(6) sites/cell). Using displacement of high-affinity 3H-dofetilide binding as a measure of interaction with the IKr channel, potencies (Ki values) for binding to the IKr channel in guinea pig myocytes for six class III antiarrhythmic agents were characterized and compared to indices of functional electrophysiologic activity in isolated guinea pig papillary muscles [EC25 values, concentration required to increase effective refractory period (ERP) 25% above baseline]. Dofetilide, E-4031, sematilide, and d-sotalol, which have been characterized previously as selective IKr blockers, displayed good agreement between Ki values for displacement of 3H-dofetilide binding (47 +/- 7 nM, 38 +/- 8 nM, 12 +/- 5 microM, and approximately 100 microM, respectively) and EC25 values for increasing ERP in papillary muscles (45.0 nM, 76.9 nM, 20.2 microM and 63.5 microM, respectively). Ibutilide and RP58866, which have been reported to act via mechanisms other than IKr block, had Ki values for displacement of 3H-dofetilide binding (16 +/- 7 nM and 17 +/- 2 nM, respectively) that were approximately 10-fold lower than EC25 values for increasing ERP in papillary muscles (185.8 nM and 223.5 nM, respectively). The potent displacement of high-affinity 3H-dofetilide binding by ibutilide and RP58866 strongly suggest a role for interaction with IKr in their actions.(ABSTRACT TRUNCATED AT 250 WORDS)

Foraging rules for nectar: food choices by painted ladies.

Butterflies selected one of two contrasting locations for 10 visits without feeding followed by 16 visits with feeding. For each visit a location provided either 35% sucrose for 20 s versus 70% sucrose for 30 s or 35% sucrose for 20 s versus 70% sucrose for 20 s. These pairings tested between choices based on rates of net energy gain, net gains per visit, and/or net gains per meal. The first pairing produced choice based on maximizing net gain per meal. Choice for the second pairing was based either on maximizing net energy gain rate or net gain per visit. The change in foraging rule was associated with a change in volume per visit for 70% sucrose Understanding choices and the evolution of nectar rewards requires detailed information on alternatives, and interpretations of foraging behavior for nectar feeders should not be based solely on a criterion of maximizing net energy gain rate during foraging.

Threshold estimation using the "chained stimuli" auditory brain stem response technique.

The chained stimuli ABR method, which allows acquisition of data for a seven-point latency-intensity function for both ears in approximately 35 min, is described. Testing of 22 ears with a simulated conductive loss, and 20 ears with sensorineural impairment, indicates that the chained stimuli method provides equivalent threshold estimates to that obtained with the conventional ABR measurement technique.