A Critical Assessment of the Golden Hour and the Impact of Procedural Timing in Stroke Thrombectomy.

BACKGROUND AND PURPOSE: Previous studies in acute ischemic stroke have demonstrated the importance of minimizing delays to endovascular treatment and keeping thrombectomy procedural times at <30-60 minutes. The purpose of this study was to investigate the impact of thrombectomy procedural times on clinical outcomes. MATERIALS AND METHODS: We retrospectively compared 319 patients having undergone thrombectomy according to procedural time (<30 minutes, 30-60 minutes, and >60 minutes) and time from stroke onset to endovascular therapy (≤6 or >6 hours). Clinical characteristics of patients with postprocedural intracranial hemorrhage were also assessed. Logistic regression was used to determine independent predictors of poor outcome at 90 days (mRS ≥3). RESULTS: Greater age (OR, 1.03; 95% CI, 1.01-1.06; P = .016), higher admission NIHSS score (OR, 1.10; 95% CI, 1.04-1.16; P = .001), history of diabetes mellitus (OR, 1.96; 95% CI, 1.05-3.65; P = .034), and postprocedural intracranial hemorrhage were independently associated with greater odds of poor outcome. Modified TICI scale scores of 2c (OR, 0.11; 95% CI, 0.04-0.28; P < .001) and 3 (OR, 0.15; 95% CI, 0.06-0.38; P < .001) were associated with reduced odds of poor outcome. Although not statistically significant on univariate analysis, onset to endovascular therapy of >6 hours was independently associated with increased odds of poor outcome (OR, 2.20; 95% CI, 1.11-4.36; P = .024) in the final multivariate model (area under the curve = 0.820). Procedural time was not independently associated with clinical outcome in the final multivariate model (P > .05). CONCLUSIONS: Thrombectomy procedural times beyond 60 minutes are associated with lower revascularization rates and worse 90-day outcomes. Procedural time itself was not an independent predictor of outcome. While stroke thrombectomy procedures should be performed rapidly, our study emphasizes the significance of achieving revascularization despite the requisite procedural time. However, the potential for revascularization must be weighed against the risks associated with multiple thrombectomy attempts.

Pipeline Diameter Significantly Impacts the Long-Term Fate of Jailed Side Branches during Treatment of Intracranial Aneurysms.

BACKGROUND AND PURPOSE: Although covered side branches typically remain patent acutely following Pipeline Embolization Device embolization of intracranial aneurysms, the long-term fate of these vessels remains uncertain. We therefore elected to investigate factors that may influence the long-term patency of these covered side branches. MATERIALS AND METHODS: We retrospectively evaluated the long-term patency of side branches covered by the Pipeline Embolization Device at our institution during treatment of intracranial aneurysms with at least 6 months of conventional angiography follow-up. Procedural and anatomic factors that might influence the fate of covered side branches were explored. RESULTS: One hundred forty-eight Pipeline Embolization Device treatments in 137 patients met the inclusion criteria. In 217 covered side branches, 29 (13.4%) were occluded on follow-up, and 40 (18.4%) were stenotic. All stenoses and occlusions were asymptomatic. In the entire cohort and in the largest subset of ophthalmic arteries, a smaller Pipeline Embolization Device diameter was associated with branch vessel occlusion (P = .001, P = .013). When we considered stenotic and occluded side branches together, smaller Pipeline Embolization Device size (P = .029) and administration of intraprocedural abciximab (P = .03) predicted side branch stenosis/occlusion, while anterior choroidal branch type (P = .003) was a predictor of gross side branch patency. CONCLUSIONS: A smaller Pipeline Embolization Device diameter is associated with delayed side branch stenosis/occlusion following Pipeline Embolization Device treatment, likely due to the higher metal density of smaller caliber devices. Although hemodynamic factors, including the potential for collateral flow, are still paramount in determining the fate of covered side branches, the amount of metal coverage at the side branch orifice also plays an important role.

Effect of central catecholamine depletion on the osmotic and nonosmotic stimulation of vasopressin (antidiuretic hormone) in the rat.

The central nervous system (CNS) mechanism(s) for the release of antidiuretic hormone (ADH) by various stimuli is unknown. In this study, the role of CNS catecholamines in effecting ADH release was examined in conscious rats 10-14 d after the cerebroventricular injection of 6-hydroxydopamine (6-OHDA). This dose of 6-OHDA caused a 67% depletion of brain tissue norepinephrine and only 3% depletion of heart norepinephrine, as compared with controls, which were injected with the vehicle buffer alone. Either intravenous 3% saline (osmotic stimulus) or intraperitoneal hyperoncotic dextran (nonosmotic stimulus) was administered to water-diuresing rats through indwelling catheters. Neither of these maneuvers changed arterial pressure, pulse, or inulin clearance in control or 6-OHDA rats. The 3% saline caused similar increases in plasma osmolality (15 mosmol/kg H(2)O) in control and 6-OHDA rats. The control rats, however, increased urinary osmolality (Uosm) to 586 mosmol/kg H(2)O, whereas 6-OHDA rats increased Uosm only to 335 mosmol/kg H(2)O (P < 0.005). These changes in Uosm were accompanied by an increase in plasma ADH to 7.6 muIU/ml in control animals vs. 2.9 muIU/ml in 6-OHDA rats (P < 0.005). All waterdiuresing animals had undetectable plasma ADH levels. Dextran-induced hypovolemia caused similar decrements (- 10%) in blood volume in both control and 6-OHDA animals, neither of which had significant changes in plasma osmolality. This nonosmotic hypovolemic stimulus caused an increase in Uosm to 753 mosmol/kg H(2)O in control rats, whereas Uosm in 6-OHDA rats increased to only 358 mosmol/kg H(2)O (P < 0.001). At the same time, ADH levels also were significantly greater in Cont rats (2.4 muIU/ml) than in the 6-OHDA animals (0.69 muIU/ml; P < 0.05). These results therefore suggest that CNS catecholamines may play an important role in mediating ADH release in response to both osmotic and nonosmotic (hypovolemic) stimuli.

The economic benefits of child safety seat misuse reduction programs and design improvements for children in rear seats.

This study finds that the break-even point for child safety seat misuse reduction programs and vehicle and seat design improvements is dollars 121 a year per child seat in use, annual misuse reduction program cost is dollars 6, and Lower Anchors and Tethers for Children (LATCH) cost dollars 13 annually per seat in use (in 2004 dollars). To estimate societal injury cost savings we compared tow-away crash outcomes for children ages 0-4, traveling in child seats in the back of passenger vehicles in 1984-1986 vs. 1999-2005. Both injury frequency and severity were compared and entered into the calculation of mean injury costs. To analyze the economic benefits of child safety seat misuse reduction programs and vehicle and seat design improvements for children sitting in rear seats of passenger vehicles, we compared outcomes of tow-away crashes for children ages 0-4 traveling in a child safety seat in two different multi-year time periods: 1984-1986 and 1999-2005. We chose 1984-1986 as a baseline as those years featured large, high-quality samples of crash data during the time period before the ongoing misuse of child seats was recognized as a public policy problem. By the early 1990s, misuse was a policy issue and misuse reduction programs were springing up.

The Efficacy of Shielding Systems for Reducing Operator Exposure during Neurointerventional Procedures: A Real-World Prospective Study.

BACKGROUND AND PURPOSE: Neurointerventional surgery may expose patients and physician operators to substantial amounts of ionizing radiation. Although strategies for reducing patient exposure have been explored in the medical literature, there has been relatively little published in regards to decreasing operator exposure. The purpose of this study was to evaluate the efficacy of shielding systems in reducing physician exposure in a modern neurointerventional practice. MATERIALS AND METHODS: Informed consent was obtained from operators for this Health Insurance Portability and Accountability Act-compliant, institutional review board-approved study. Operator radiation exposure was prospectively measured during 60 consecutive neurointerventional procedures from October to November 2013 using a 3-part lead shielding system. Exposure was then evaluated without lead shielding in a second 60-procedure block from April to May 2014. A radiation protection drape was randomly selected for use in half of the cases in each block. Two-way analysis of covariance was performed to test the effect of shielding systems on operator exposure while controlling for other covariates, including procedure dose-area product. RESULTS: Mean operator procedure dose was 20.6 µSv for the entire cohort and 17.7 µSv when using some type of shielding. Operator exposure significantly correlated with procedure dose-area product, but not with other covariates. After we adjusted for procedure dose-area product, the use of lead shielding or a radiation protection drape significantly reduced operator exposure by 45% (F = 12.54, P < .0001) and 29% (F = 7.02, P = .009), respectively. The difference in protection afforded by these systems was not statistically significant (P = .46), and their adjunctive use did not provide additional protection. CONCLUSIONS: Extensive lead shielding should be used as much as possible in neurointerventional surgery to reduce operator radiation exposure to acceptable levels. A radiation protection drape is a reasonable alternative when standard lead shielding is unavailable or impractical to use without neglecting strategies to minimize the dose.

Use of an inverse agonist radioligand [3H]A-317920 reveals distinct pharmacological profiles of the rat histamine H3 receptor.

Selective radioligands for histamine H(3) receptors have been used to characterize H(3) receptor pharmacology by radioligand binding assays and to determine H(3) receptor distribution by tissue autoradiography. Here we report the synthesis and receptor binding characterization of [(3)H]A-317920 (furan-2-carboxylic acid(2-[4-[3-([3,5-(3)H]4-cyclopropanecarbonyl-phenoxy)-propyl]-piperazin-1-yl]-1-methyl-2-oxo-ethyl)-amide), a high affinity inverse agonist radioligand for the rat H(3) receptor. The binding of [(3)H]A-317920 to rat cortical and cloned H(3) receptors revealed fast on- and slower off-rate kinetics with calculated K(d) values in agreement with those determined in saturation binding assays (0.2 nM for both receptors). Further, we compared [(3)H]A-317920 with the agonist [(3)H](N)-alpha-methylhistamine ([(3)H]NalphaMH) as radioligand tools to study receptor pharmacology. Agonists and antagonists displaced [(3)H]NalphaMH with one-site binding characteristics and Hill slopes approached unity. In contrast, although antagonists exhibited one-site binding, [(3)H]A-317920 displacement by agonists was best fit by two-site binding models, and the potencies of the high affinity, GDP-sensitive sites correlated with the potencies defined in [(3)H]NalphaMH binding. Unlike [(125)I]iodoproxyfan, [(3)H]A-317920 exhibits potent and selective binding to rat H(3) receptors with low binding to non-H(3) sites, including cytochrome P450. These findings show that [(3)H]A-317920 is a potent rat H(3) receptor antagonist radioligand and has utility for studying H(3) receptor pharmacology.

The three-dimensional scintillation dosimetry method: test for a 106Ru eye plaque applicator.

The need for fast, accurate and high resolution dosimetric quality assurance in radiation therapy has been outpacing the development of new and improved 2D and 3D dosimetry techniques. This paper summarizes the efforts to create a novel and potentially very fast, 3D dosimetry method based on the observation of scintillation light from an irradiated liquid scintillator volume serving simultaneously as a phantom material and as a dose detector medium. The method, named three-dimensional scintillation dosimetry (3DSD), uses visible light images of the liquid scintillator volume at multiple angles and applies a tomographic algorithm to a series of these images to reconstruct the scintillation light emission density in each voxel of the volume. It is based on the hypothesis that with careful design and data processing, one can achieve acceptable proportionality between the local light emission density and the locally absorbed dose. The method is applied to a Ru-106 eye plaque immersed in a 16.4 cm3 liquid scintillator volume and the reconstructed 3D dose map is compared along selected profiles and planes with radiochromic film and diode measurements. The comparison indicates that the 3DSD method agrees, within 25% for most points or within approximately 2 mm distance to agreement, with the relative radiochromic film and diode dose distributions in a small (approximately 4.5 mm high and approximately 12 mm diameter) volume in the unobstructed, high gradient dose region outside the edge of the plaque. For a comparison, the reproducibility of the radiochromic film results for our measurements ranges from 10 to 15% within this volume. At present, the 3DSD method is not accurate close to the edge of the plaque, and further than approximately 10 mm (<10% central axis depth dose) from the plaque surface. Improvement strategies, considered important to provide a more accurate quick check of the dose profiles in 3D for brachytherapy applicators, are discussed.

Isolated cardiac aspergillosis after bone marrow transplantation.

A 15-year-old girl developed isolated cardiac aspergillosis after allogeneic bone marrow transplantation. Predisposing factors included severe graft-vs-host disease with gastrointestinal tract involvement and immunosuppressive therapy including cyclosporine. The patient died of cardiopulmonary failure 11 weeks after transplantation.

Relationship between passive tissue strain and collagen uncoiling during healing of infarcted myocardium.

OBJECTIVE: The structure of the collagen scar during healing of a myocardial infarction is a determinant of the function of the remodeled tissue. We hypothesize that the passive deformations of both scar and normal tissue are related to the underlying collagen uncoiling as the tissue stretches, and that the unloaded tortuosity of the collagen may be a determinant of tissue stiffness at low ventricular pressure. Hence collagen uncoiling and tissue strain were measured during passive loading in normal tissue, and in healing infarct tissue. METHODS: Left ventricles of rats were infarcted by ligation of the left anterior descending artery for 2 weeks. Surface strains were measured during passive inflation in the scar region in one set of excised hearts, and other arrested hearts were fixed at different ventricular pressures, after which collagen tortuosity was measured in the infarcted and normal tissue. RESULTS: Passive loading strains were smaller in the scar in both the fiber and cross-fiber directions. Tortuosity decreased with load in normal and infarcted tissue, with fibrils tending to straighten more in the scar tissue at higher pressures (1.056 +/- 0.009 vs. 1.024 +/- 0.009 at P = 20 mmHg) with similar tortuosities at zero pressure (1.110 +/- 0.012 vs. 1.098 +/- 0.019). The decrease in tortuosity with strain was greater for the infarcted tissue. CONCLUSIONS: The greater stiffness of infarcted tissue at low pressure is not due to 'straightened' collagen fibers, and there may be a different three-dimensional structure of infarct vs. normal coiled collagen fibers which can affect the material properties of these tissues.

Reversible Cerebral Vasoconstriction Syndrome, Part 2: Diagnostic Work-Up, Imaging Evaluation, and Differential Diagnosis.

The diagnostic evaluation of a patient with reversible cerebral vasoconstriction syndrome integrates clinical, laboratory, and radiologic findings. Imaging plays an important role by confirming the presence of cerebral vasoconstriction; monitoring potential complications such as ischemic stroke; and suggesting alternative diagnoses, including CNS vasculitis and aneurysmal subarachnoid hemorrhage. Noninvasive vascular imaging, including transcranial Doppler sonography and MR angiography, has played an increasingly important role in this regard, though conventional angiography remains the criterion standard for the evaluation of cerebral artery vasoconstriction. Newer imaging techniques, including high-resolution vessel wall imaging, may help in the future to better discriminate reversible cerebral vasoconstriction syndrome from primary angiitis of the CNS, an important clinical distinction.

Reversible Cerebral Vasoconstriction Syndrome, Part 1: Epidemiology, Pathogenesis, and Clinical Course.

Reversible cerebral vasoconstriction syndrome is a clinical and radiologic syndrome that represents a common presentation of a diverse group of disorders. The syndrome is characterized by thunderclap headache and reversible vasoconstriction of cerebral arteries, which can either be spontaneous or related to an exogenous trigger. The pathophysiology of reversible cerebral vasoconstriction syndrome is unknown, though alterations in cerebral vascular tone are thought to be a key underlying mechanism. The syndrome typically follows a benign course; however, reversible cerebral vasoconstriction syndrome may result in permanent disability or death in a small minority of patients secondary to complications such as ischemic stroke or intracranial hemorrhage.

Pathological tumor-node-metastasis (pTNM) staging for papillary and follicular thyroid carcinomas: a retrospective analysis of 700 patients.

The TNM classification (tumor-node-metastasis) was adopted by the American Joint Committee on Cancer and the International Union against Cancer a decade ago to avoid heterogeneity of prognostic classification schemes used for differentiated thyroid cancers. To date, however, clinical data based on this classification are lacking. We retrospectively evaluate the prognosis of 700 patients (208 men and 492 women) with papillary (89%) and follicular (11%) thyroid cancers according to the pathological TNM (pTNM) staging system, treated over a 25-yr period (1970-1995). Patients who received primary treatment at our center constituted 87.4% of the cases; the majority underwent total thyroidectomy, followed by 131I ablative therapy in high risk groups, as standard treatment. Clinical and follow-up data were obtained from the medical records and our cancer registry. Disease-free and cancer-specific survival data were analyzed by Kaplan-Meier product limit estimates and Cox proportional hazard models. Patient distribution by the pTNM system were: stage I, 516 patients; stage II, 57 patients; stage III, 104 patients; and stage IV, 23 patients. Over a mean +/- SE follow-up of 11.3 +/- 0.3 yr, the overall cancer recurrence and mortality rates were 20.5% and 8.4%, respectively. However, the respective cancer recurrence and mortality rates were distinctly different in the various pTNM stages: 15.4% and 1.7% in stage I, 22% and 15.8% in stage II, 46.4% and 30% in stage III, and 66.7% and 60.9% in stage IV tumors. Using actuarial survival plots, a clear separation in both disease-free survival and cancer-specific survival was noted among all the stages (P < 0.0001). Risk factors analyses showed a significant association between all the prognostic variables used in TNM staging (age, tumor size, extent of primary tumor, and presence of nodal or distant metastases) and the observed end points of recurrence or death from thyroid cancer. After correcting for TNM stages, the risk of cancer recurrence was halved in female compared to male patients, whereas this was 1.7-fold higher in multifocal than unifocal tumors. Conversely, cancer mortality was 3.4-fold higher in follicular than papillary thyroid cancer. In the analysis of effect of primary treatment among 492 patients with tumor more advanced than the T1N0M0 category, patients who underwent less extensive surgery (lobectomy or subtotal thyroidectomy) had a 2.5-fold risk of cancer recurrence (P < 0.0001) and a 2.2-fold risk of death (P < 0.01) compared to those who underwent total or near-total thyroidectomy. Patients not treated with 131I ablation had a 2.1-fold greater risk of cancer recurrence (P < 0.0001) than those given 131I ablation, although no difference was noted in deaths from thyroid cancer. Based on our data, the pTNM classification is useful in distinguishing patients with different prognostic outcomes. However, the small patient numbers in pTNM stages other than stages I precludes us from evaluating its usefulness as a guide for therapy. Until prospective data could be accrued from controlled treatment trials, we support the standard practice of total thyroidectomy followed by 131I ablative therapy (if focal iodide uptake was noted) in patients with papillary thyroid cancer more advanced than the T1N0M0 category or of multicentric nature and in the majority of patients with follicular thyroid cancer.

Influence of lymphocytic thyroiditis on the prognostic outcome of patients with papillary thyroid carcinoma.

Both the association between lymphocytic thyroiditis (LT) and papillary thyroid carcinoma (PTC), and the prognostic significance of lymphocytic infiltrate in patients with thyroid malignancy, remain controversial. We examine the above relationships by retrospectively reviewing our series of patients treated for differentiated nonmedullary thyroid carcinoma at University of California-San Francisco over a 25-yr period (1970-1995). Of the 631 patients with complete data for analysis, 128 patients (20.3%) showed concomitant histologic evidence of LT and 503 patients (79.7%) had no evidence of LT. Prognostic outcome was assessed using Kaplan-Meier survival plots and analysis of risk factors by Cox's proportional-hazard modeling. The cohort with LT revealed a higher frequency of PTC (97.7% vs. 87.3%) and female patients (85.2% vs. 66.8%), a lower frequency of extrathyroidal invasion (7.8% vs. 23.3%) and nodal metastases (25.8% vs. 43.3%), and absence of distant metastases (0% vs. 4.8%), respectively, compared with those without LT. At initial surgery, a significantly greater proportion of patients with LT belonged to lower pathological tumor-node-metastasis stages, compared with those without LT (stage 1, 86.7% vs. 73%; stage 2, 4.7% vs. 8.3%; stage 3, 8.6% vs. 15.3%; and stage 4, 0% vs. 3.4%). Over a mean +/- SE follow-up period of 11.1 +/- 0.4 yr, patients with LT had significantly lower cancer recurrence rate (6.3% vs. 24.1%; P < 0.0001) and cancer mortality rate (0.8% vs. 8.0%; P = 0.001), respectively, compared with those without LT. In summary, our series showed a relatively common occurrence of LT in patients with PTC, and we believed that lymphocytic infiltration developed mainly in response to the tumor itself. We also found a more favorable course of PTC in the presence of LT; this supports the hypothesis that lymphocytic infiltration represents a form of immune reaction to control tumor growth and proliferation.

Fine needle aspiration biopsy. Has its time come?

Fine needle aspiration biopsy has been undergoing a revival in the United States, but a recent survey showed that many clinicians were uncertain how to utilize this test. The scope of fine needle aspiration biopsy as practiced at the University of California, San Francisco, and the San Francisco General Hospital is described. The relevant accuracy data available in the world literature on this procedure are also reviewed.

Lack of correlation of clinical breast examination with high-risk histopathology.

PURPOSE: Routine breast examination frequently finds differences in palpable density and palpable nodularity, but it is not known if these differences correlate with the presence of high-risk histopathology. PATIENTS AND METHODS: To test for a relationship between clinical breast examination and histopathology, we devised separate, 4-point scales of clinical density and nodularity and validated these scales by repeat examinations 4 or more months apart in 199 separate breasts (the scale was the same or within 1 point on repeat examination 87% of the time for density and 90% for nodularity). We then used these two clinical scales to compare density and nodularity to histopathology of breast tissue at the margins of segmental resections in 60 women undergoing breast-conserving treatment of primary breast cancer. In cases such as these, a large sample of "normal" tissue is intentionally removed when the wide excision is done to obtain negative margins. Histopathology at the margins was graded according to the consensus panel of the American College of Pathology; as might be expected in women with previous cancer, some higher-risk histopathology was found in 37% of cases. A relationship was sought using Spearman's rank correlation coefficient. RESULTS: Neither clinical breast density (rho = 0.16) nor clinical breast nodularity (rho = 0.01) related to the presence of high-risk histopathology in the underlying tissue. Interestingly, breast nodularity increased with age (rho = 0.28), and clinical density and nodularity were inversely related (rho = -0.28). CONCLUSION: We conclude that neither clinical breast density nor nodularity correlates with histopathology and that it is unlikely that a larger study would find a clinically useful correlation. Therefore, a clinical examination should not be used to decide that high-risk histopathology is likely to be present in an individual woman's breast.

Boc-CCK-4 derivatives containing side-chain ureas as potent and selective CCK-a receptor agonists.

Novel Boc-CCK-4 derivatives were communicated recently as having high potency and selectivity for the CCK-A receptor (Shiosaki et al. J. Med. Chem. 1990, 33, 2950-2952). While Boc-CCK-4 binds selectively to the CCK-B receptor, replacement of the methionine with an N epsilon-substituted lysine dramatically reversed receptor selectivity, leading to the development of this novel series of tetrapeptides. A detailed structure-activity analysis of a series of urea-substituted tetrapeptides, represented by the general structure Boc-Trp-Lys(N epsilon-CO-NHR)-Asp-Phe-NH2, revealed that a number of substituted phenyl, naphthyl, and aliphatic urea residues in the lysine side chain yielded potent and selective CCK-A ligands. These tetrapeptides elicit full agonist responses in stimulating pancreatic amylase release that are effectively blocked by a selective CCK-A receptor antagonist. Conversion of the urea to a thiourea significantly reduced CCK-A binding potency as did replacement of the lysine with the homologous ornithine or homolysine. Tetrapeptides that were partial agonists (less than 80% efficacy) in phosphoinositide (PI) hydrolysis relative to CCK-8 did not exhibit high-dose inhibition of amylase secretion in guinea pig acini.

Development of potent and selective CCK-A receptor agonists from Boc-CCK-4: tetrapeptides containing Lys(N epsilon)-amide residues.

A series of Boc-CCK-4 derivatives represented by the general structure Boc-Trp-Lys(N epsilon-COR)-Asp-Phe-NH2, where R is an aromatic, heterocyclic, or aliphatic group, are potent and selective CCK-A receptor agonists. These amide-bearing compounds complement the previously described urea-based tetrapeptides (Shiosaki et al. J. Med. Chem. 1991, 34, 2837-2842); structure-activity studies revealed parallel as well as divergent trends between these two series. A significant correlation was observed between pancreatic binding affinity and the resonance constant R of the phenyl substituent in one particular series of derivatives. Sulfation of phenolic amides appended onto the epsilon-amino group of the lysine did not affect affinity for the CCK-A receptor in contrast to the 500-fold increase in binding potency observed upon sulfation of CCK-8, suggesting that the lysine appendage and the sulfated tyrosine in CCK-8, both key structural elements that impart high affinity for the CCK-A receptor, are interacting differently with the receptor. The amide-bearing tetrapeptides are full agonists relative to CCK-8 in stimulating pancreatic amylase release while being partial agonists in eliciting phosphoinositide (PI) hydrolysis. Both effects were blocked by selective CCK-A receptor antagonists.

Synthesis and biological activity of CCK heptapeptide analogues. Effects of conformational constraints and standard modifications on receptor subtype selectivity, functional activity in vitro, and appetite suppression in vivo.

A series of modifications of the CCK7 analogue (des-NH2)Tyr(SO3-)-Nle-Gly-Trp-Nle-Asp-Phe-NH2 was prepared and tested for binding to guinea pig CCK-A and CCK-B receptors and in CCK-A-mediated functional assays. Selected analogues also were tested for appetite suppressant activity in rats. Several conformationally restricted residues in the C-terminal tetrapeptide region, including delta Z-Phe33, (N-Me)Phe33, (N-Me)Asp32, (N-Me)Leu31, and 3PP31 (3PP = trans-3-n-propyl-L- proline) were found to be acceptable modifications at one or both receptor subtypes. The (N-Me)Asp32 and (N-Me)Leu31 modifications afforded potent and selective CCK-A and CCK-B ligands, respectively. SAR studies in the N-terminal acyldipeptide region examined structural requirements for the side chain at position 28, where Gly and Pro replacements were found to possess high affinity at both receptor subtypes. Other conformationally restrictive modifications were less active. All of the analogues that showed high affinity (less than 10 nM) for the CCK-A receptor also were full agonists in amylase release and most were full or nearly full agonists in the phosphoinositide (PI) turnover assay, the most notable exception being the delta Z-Phe33 analogue, which showed 69% of the maximal response in the PI assay. Potent activity in suppression of food intake in rats was found for selected analogues.

Novel Asp32-replacement tetrapeptide analogues as potent and selective CCK-A agonists.

A series of novel CCK tetrapeptide analogues of the general formula Boc-Trp-Lys(Tac)-N(R)-(CH2)nCON(R')Phe-NH2 (Tac = o-tolylaminocarbonyl), where R,R' = H or Me and n = 1-5, have been synthesized and tested. These analogues, which lack an acidic residue at the penultimate position, demonstrated surprisingly high CCK-A receptor affinity and selectivity. The effect of N-methylation pattern on CCK-A receptor affinity showed consistent trends for analogues in which n = 1, 2, or 3, with the di-N-methylated analogues having the highest affinity in each case. However, none of these analogues had full agonist activity, as measured by percent maximal PI hydrolysis. Two conformationally constrained analogues also demonstrated high CCK-A receptor affinity and selectivity, as well as nearly maximal agonist activity. In addition, one of these conformationally-constrained analogues demonstrated anorectic activity in rats.

CCK-A-selective tetrapeptides containing lys(N epsilon)-amide residues: favorable in vivo and in vitro effects of N-methylation at the aspartyl residue.

Previous structure-activity studies on a series of CCK-A selective tetrapeptide agonists, typified by A-71623 (Boc-Trp-Lys(CONH-Ph-o-Me)-Asp-(N-Me)Phe-NH2), have shown that replacement of the Lys(N epsilon-carbamoyl) substituent with N epsilon-acyl substituents resulted in partial agonists with moderate to high affinities for the CCK-A receptor and that replacement of the C-terminal dipeptide with either (N-Me)Asp-Phe or (N-Me)Asp-(N-Me)Phe was highly favorable to in vitro and in vivo CCK activity. The present study demonstrates that although analogues in the epsilon-amide series that are N-methylated at the Phe position are weakly active or inactive in an in vivo rat appetite suppression assay, incorporation of (N-Me)Asp or (N-Me)Asp-(N-Me)Phe modifications in this series results in analogues with markedly improved in vivo activity. In in vitro assays, there is minimal effect of N-methylation pattern on binding affinity, whereas there is a trend toward improved functional activity in the phosphatidylinositol hydrolysis assay in analogues containing (N-Me)Asp.