Measured proton electromagnetic structure deviates from theoretical predictions.

The visible world is founded on the proton, the only composite building block of matter that is stable in nature. Consequently, understanding the formation of matter relies on explaining the dynamics and the properties of the proton's bound state. A fundamental property of the proton involves the response of the system to an external electromagnetic field. It is characterized by the electromagnetic polarizabilities1 that describe how easily the charge and magnetization distributions inside the system are distorted by the electromagnetic field. Moreover, the generalized polarizabilities2 map out the resulting deformation of the densities in a proton subject to an electromagnetic field. They disclose essential information about the underlying system dynamics and provide a key for decoding the proton structure in terms of the theory of the strong interaction that binds its elementary quark and gluon constituents. Of particular interest is a puzzle in the electric generalized polarizability of the proton that remains unresolved for two decades2. Here we report measurements of the proton's electromagnetic generalized polarizabilities at low four-momentum transfer squared. We show evidence of an anomaly to the behaviour of the proton's electric generalized polarizability that contradicts the predictions of nuclear theory and derive its signature in the spatial distribution of the induced polarization in the proton. The reported measurements suggest the presence of a new, not-yet-understood dynamical mechanism in the proton and present notable challenges to the nuclear theory.

Seroepidemiological Studies of Arboviruses in Africa.

The literature on sero-epidemiological studies of flaviviral infections in the African continent is quite scarce. Much of the viral epidemiology studies have been focussing on diseases such as HIV/AIDS because of their sheer magnitude and impact on the lives of people in the various affected countries. Increasingly disease outbreaks caused by arboviruses such as the recent cases of chikungunya virus, dengue virus and yellow fever virus have prompted renewed interest in studying these viruses. International agencies from the US, several EU nations and China are starting to build collaborations to build capacity in many African countries together with established institutions to conduct these studies. The Tofo Advanced Study Week (TASW) was established to bring the best scientists from the world to the tiny seaside town of Praia do Tofo to rub shoulders with African virologists and discuss cutting-edge science and listen to the work of researchers in the field. In 2015 the 1st TASW focussed on Ebola virus. The collections of abstracts from participants at the 2nd TASW which focused on Dengue and Zika virus as well as presentations on other arboviruses are collated in this chapter.

A phase IIb, open-label, randomized controlled dose ranging multi-centre trial to evaluate the safety, tolerability, pharmacokinetics and exposure-response relationship of different doses of delpazolid in combination with bedaquiline delamanid moxifloxacin in adult subjects with newly diagnosed, uncomplicated, smear-positive, drug-sensitive pulmonary tuberculosis.

BACKGROUND: Linezolid is an effective, but toxic anti-tuberculosis drug that is currently recommended for the treatment of drug-resistant tuberculosis. Improved oxazolidinones should have a better safety profile, while preserving efficacy. Delpazolid is a novel oxazolidinone developed by LegoChem Biosciences Inc. that has been evaluated up to phase 2a clinical trials. Since oxazolidinone toxicity can occur late in treatment, LegoChem Biosciences Inc. and the PanACEA Consortium designed DECODE to be an innovative dose-ranging study with long-term follow-up for determining the exposure-response and exposure-toxicity relationship of delpazolid to support dose selection for later studies. Delpazolid is administered in combination with bedaquiline, delamanid and moxifloxacin. METHODS: Seventy-five participants with drug-sensitive, pulmonary tuberculosis will receive bedaquiline, delamanid and moxifloxacin, and will be randomized to delpazolid dosages of 0 mg, 400 mg, 800 mg, 1200 mg once daily, or 800 mg twice daily, for 16 weeks. The primary efficacy endpoint will be the rate of decline of bacterial load on treatment, measured by MGIT liquid culture time to detection from weekly sputum cultures. The primary safety endpoint will be the proportion of oxazolidinone class toxicities; neuropathy, myelosuppression, or tyramine pressor response. Participants who convert to negative liquid media culture by week 8 will stop treatment after the end of their 16-week course and will be observed for relapse until week 52. Participants who do not convert to negative culture will receive continuation phase treatment with rifampicin and isoniazid to complete a six-month treatment course. DISCUSSION: DECODE is an innovative dose-finding trial, designed to support exposure-response modelling for safe and effective dose selection. The trial design allows assessment of occurrence of late toxicities as observed with linezolid, which is necessary in clinical evaluation of novel oxazolidinones. The primary efficacy endpoint is the change in bacterial load, an endpoint conventionally used in shorter dose-finding trials. Long-term follow-up after shortened treatment is possible through a safety rule excluding slow-and non-responders from potentially poorly performing dosages. TRIAL REGISTRATION: DECODE was registered in ClinicalTrials.gov before recruitment start on 22 October 2021 (NCT04550832).

Pharmacology and clinical use of sex steroid hormone receptor modulators.

Sex steroid receptors are ligand-triggered transcription factors. Oestrogen, progesterone and androgen receptors form, together with the glucocorticoid and mineralocorticoid receptors, a subgroup of the superfamily of nuclear receptors. They share a common mode of action, namely translating a hormone-i.e. a small-molecule signal-from outside to changes in gene expression and cell fate, and thereby represent "natural" pharmacological targets.For pharmacological therapy, these receptors have originally been addressed by hormones and synthetic hormone analogues in order to overcome pathologies related to deficiencies in the natural ligands. Another major use for female sex hormone receptor modulators is oral contraception, i.e. birth control.On the other side, blocking the activity of sex steroid receptors has become an established way to treat hormone-dependent malignancies, such as breast and prostate cancer.In this review, we will discuss how the experience gained from the classical pharmacology of these receptors and their molecular similarities led to new options for the treatment of gender-specific diseases and highlight recent progress in medicinal chemistry of sex hormone-modulating drugs.

Diagnosis of paediatric TB using Xpert® MTB/RIF Ultra on fresh respiratory samples.

OBJECTIVE: To evaluate the diagnostic accuracy of Xpert® MTB/RIF Ultra (Ultra) on fresh respiratory samples for the diagnosis of pulmonary TB (PTB) in children.METHODS: Between July 2017 and December 2019, children with presumed TB were prospectively enrolled at clinical sites in three African countries. Children were assessed using history, physical examination and chest X-ray. Sputum or gastric aspirate samples were analysed using Ultra and culture. The diagnostic accuracy of Ultra was calculated against culture as the reference standard.RESULTS: In total, 547children were included. The median age was 4.7 years, 77 (14.1%) were HIV infected and 77 (14.1%) had bacteriologically confirmed TB. Ultra detected an additional 20 cases in the group of children with negative culture results. The sensitivity of Ultra was 66.3% (95% CI 47-82), and the specificity was 95.4% (95% CI 89-99) when assessed against culture as the reference standard.CONCLUSION: Despite the improved performance of Ultra as compared to Xpert as was previously reported, its sensitivity remains sub-optimal for the detection of TB in children. Ultra detected additional 20 cases which otherwise could not have been detected by culture alone, suggesting that the latter is an imperfect reference standard.

Successful management of an MRSA outbreak in a neonatal intensive care unit.

We report an MRSA outbreak in our 25-bed tertiary neonatal intensive care unit (NICU), which was successfully contained. Methods include a retrospective review of patient files, microbiology records and meeting protocols. During the seven months of outbreak, 27 patients and seven health care workers (HCWs) had positive cultures for MRSA. The outbreak was caused by the epidemic Rhine-Hessen strain; cultured isolates were monoclonal. After a sharp increase of the number of new MRSA-cases the installation of an outbreak management team (OMT) and implementation of comprehensive measures (extensive screening and decolonization strategy including orally applied vancomycin, isolation wards, intensive disinfection regimen) successfully terminated the outbreak within one month. Ten (53%) of 19 patients with completed follow-up and all of the HCWs were decolonized successfully. Gastrointestinal colonization was present in 15 of 27 (56%) neonates, and was associated with poor decolonization success (30% vs. 78% in absence of gastrointestinal colonization). A comprehensive outbreak management can terminate an outbreak in a NICU setting within a short time. Thorough screening of nares, throat and especially stool is necessary for correct cohorting. Gastrointestinal decolonization in neonates seems difficult.

Achieving signalling selectivity of ligands for the corticotropin-releasing factor type 1 receptor by modifying the agonist's signalling domain.

BACKGROUND AND PURPOSE: Most of the pharmaceuticals target G-protein-coupled receptors (GPCRs) which can generally activate different signalling events. The aim of this study was to achieve functional selectivity of corticotropin-releasing factor receptor type 1 (CRF(1)) ligands. EXPERIMENTAL APPROACH: We systematically substituted urocortin, a natural peptide agonist of CRF(1), with bulky amino acids (benzoyl-phenylalanine, naphthylalanine) and determined the effect of the analogues on coupling of CRF(1) to Gs- and Gi-protein in human embryonic kidney cells, using receptor binding, [(35)S]-GTPgammaS binding stimulation, and cAMP accumulation assays. KEY RESULTS: Native ligands stimulated Gs and Gi activation through CRF(1), resulting in stimulation and then inhibition of cAMP accumulation. Single replacements in urocortin at positions 6-15 led, dependent on the position and nature of the substituent, to ligands that conserved Gs activity, but were devoid of Gi activity, only stimulating cAMP accumulation, and competitively antagonized the Gi activation by sauvagine. In contrast, analogues with substitutions outside this sequence non-selectively activated Gs and Gi, as urocortin did. CONCLUSIONS AND IMPLICATIONS: Modifications in a specific region, which we have called the signalling domain, in the polypeptide agonist urocortin resulted in analogues that behaved as agonists and, at the same time, antagonists for the activation of different G-proteins by CRF(1). This finding implies significant differences between active conformations of the receptor when coupled to different G-proteins. A similar structural encoding of signalling information in other polypeptide hormone receptor ligands would result in a general concept for the development of signalling-selective drug candidates.

Identifying children with tuberculosis among household contacts in The Gambia.

SETTING: Greater Banjul Area of the Gambia. OBJECTIVES: To identify co-prevalent tuberculosis (TB) among child contacts of adults with smear-positive TB. DESIGN: Child contacts aged <15 years in the immediate household and compound were prospectively enrolled and evaluated for TB disease using screening questionnaires and the tuberculin skin test (TST). Symptomatic and/or TST-positive (10 mm) contacts were further investigated. RESULTS: Of 4042 child contacts who underwent symptom screening and TST, 3339 (82.6%) were diagnosed as TB-exposed but not infected, 639 (15.8%) were latently infected and 64 (1.6%) had co-prevalent TB. Of the 64 TB cases, 50 (78.1%) were from within the immediate household of the index case, and 14 (21.9%) from within the same compound. Of the 27 asymptomatic but TST-positive children diagnosed with TB, 7 were microbiologically confirmed. The median age of the TB cases was 4.4 years (interquartile range 1.9-6.9); 53.1% were aged <5 years. Of the 4042 child contacts, 206 (5%) slept in the same bed as the index case; 28.1% of all TB cases occurred in this group. Symptom screening alone would have detected only 57.8% of the co-prevalent cases. CONCLUSION: In our community setting, if contact tracing is restricted to symptom screening and immediate households only, nearly half of all co-prevalent TB disease in child contacts would be missed.

Evidence that corticotropin-releasing factor receptor type 1 couples to Gs- and Gi-proteins through different conformations of its J-domain.

BACKGROUND AND PURPOSE: According to the two-domain model for the corticotropin-releasing factor receptor type 1 (CRF(1)), peptide antagonists bind to the N-terminal domain (N-domain), non-peptide antagonists to the transmembrane region (J-domain), whereas peptide agonists attach to both the N- and J-domain of the receptor to express activity. The aim of this study was to search for possible differences in the antagonism of the Gs- and Gi-protein coupling of CRF(1) by a peptide (alpha-helical CRF(9-41)) and non-peptide antagonist (antalarmin), to determine whether the conformational requirements of the activated CRF(1) states for Gs and Gi coupling are similar or different. EXPERIMENTAL APPROACH: We studied the inhibitory effect of alpha-helical CRF(9-41) and antalarmin on the coupling of CRF(1) to Gs- and Gi-protein in human embryonic kidney cells, using the [(35)S]-GTPgammaS binding stimulation assay. KEY RESULTS: The non-peptide antagonized the receptor coupling to Gs competitively but that to Gi noncompetitively, and its antagonistic potency was different for urocortin- and sauvagine-evoked G-protein activation. In contrast, the peptide antagonist exhibited uniformly competitive antagonism. CONCLUSIONS AND IMPLICATIONS: The results allow us to extend the two-domain model of CRF(1) activation by assuming that CRF(1) agonists activate the receptor by binding to at least two ensembles of J-domain configurations which couple to Gs or Gi, that are in turn antagonized by a non-peptide antagonist competitively and allosterically, respectively. It is further concluded that the allosteric mechanism of non-peptide antagonism is not valid for the Gs-mediated physiological activities of CRF(1).

Isoniazid preventive treatment among child contacts of adults with smear-positive tuberculosis in The Gambia.

Setting: Greater Banjul area of The Gambia. Objectives: To evaluate uptake, adherence and completion of treatment among tuberculosis (TB) exposed children in The Gambia when isoniazid preventive treatment (IPT) is delivered at home Design: Child (age <5 years) contacts of adults with smear-positive TB were prospectively enrolled. Following symptom screening, tuberculin skin testing and clinical evaluation where indicated, those without disease were placed on daily isoniazid, provided monthly at home. Adherence was assessed by pill counts and IsoScreen™ urine test. Results: Of 404 contacts aged <5 years, 368 (91.1%) were offered IPT. Of the 328 (89.4%) for whom consent was received and who commenced IPT, 18 (5.5%) dropped out and 310 (94.5%) remained on IPT to the end of the 6-month regimen. Altogether, 255/328 children (77.7%, 95%CI 73.2-82.2) completed all 6 months, with good adherence. The IsoScreen test was positive in 85.3% (435/510) of all tests among those defined as having good adherence by pill count and in 16% (8/50) of those defined as having poor adherence (P < 0.001). A cascade of care analysis showed an overall completion rate with good adherence of 61% for all child contacts. Conclusion: Home-delivered IPT among child contacts of adults with smear-positive TB in The Gambia achieved verifiable high uptake and adherence rates. System rather than patient factors are likely to determine the success of IPT at national level.

Contexte : Région du Grand Banjul en Gambie.Objectifs : Evaluer la couverture, l'adhésion et l'achèvement du traitement parmi des enfants exposés à la tuberculose (TB) en Gambie quand le traitement préventif par isoniazide (TPI) est donné à domicile.Schéma : Les enfants âgés de <5 ans, contacts d'adultes atteints de TB à frottis positif, ont été enrôlés de manière prospective. Après dépistage sur les symptômes, test cutané à la tuberculine et évaluation clinique quand cela était indiqué, les enfants non malades ont été mis sous isoniazide, fourni une fois par mois à domicile. L'adhésion a été évaluée par un comptage des comprimés et par un test urinaire IsoScreen™.Résultats : Sur 404 contacts âgés de <5 ans, 368 (91,1%) ont été invités à bénéficier du TPI, et 328 (89,4%) ont consenti et commencé le TPI. Sur ces 328 enfants, 18 (5,5%) ont abandonné et 310 (94,5%) sont restés sous TPI jusqu'à la fin du 6e mois. Au total, 255/328 enfants (77,7% ; IC95% 73,2­82,2) ont achevé les 6 mois de traitement avec une bonne adhésion. Le test IsoScreen a été positif chez 85,3% (435/510) de tous les tests parmi ceux définis comme ayant une bonne adhésion par le comptage des comprimés et chez 16% (8/50) de ceux définis comme ayant une adhésion médiocre (P < 0,001). L'analyse de la « cascade des soins ¼ a montré, pour tous les enfants contacts, un taux de bonne adhésion d'ensemble de 61%.Conclusion: L'administration à domicile du TPI à des enfants contacts d'adultes atteints de TB à frottis positif en Gambie a abouti à une bonne couverture et à un bon taux d'adhésion, tous deux vérifiables. Ce sont les facteurs de système plutôt que ceux liés au patient qui sont susceptibles de déterminer le succès du TPI au niveau national.

Marco de referencia: La zona del Gran Banjul en Gambia.Objetivos: Evaluar la aceptación del tratamiento preventivo con isoniazida (TPI), su cumplimiento y su compleción por parte de los niños expuestos en Gambia, cuando se suministra el tratamiento en los hogares.Método: Se incluyeron en el estudio de manera prospectiva los niños menores de 5 años de edad que eran contactos de un adulto con diagnóstico de tuberculosis (TB) y baciloscopia positiva. Luego de la detección sistemática a partir de los síntomas, se practicaron la prueba cutánea de la tuberculina y la evaluación clínica cuando estaban indicadas; en caso de ausencia de enfermedad activa se inició el tratamiento diario con isoniazida, la cual se suministraba en el hogar cada mes. Se evaluó el cumplimiento en función del recuento de los comprimidos y la prueba IsoScreen™ en muestras de orina.Resultados: En los 404 contactos menores de 5 años de edad, se ofreció el TPI a 368 niños (91,1%) y 328 lo aceptaron y comenzaron a recibirlo (89,4%). De este grupo, 18 niños abandonaron el tratamiento (5,5%) y 310 recibían aun el medicamento al final del 6 mes (94,5%). De los 328 niños, 255 terminaron los 6 meses de tratamiento, con un cumplimiento satisfactorio (77,7%; IC del 95% de 73,2 hasta 82,2). La prueba IsoScreen fue positiva en el 85,3% (435/510) de los casos definidos con cumplimiento adecuado según el recuento de comprimidos y en el 16% (8/50) de los casos cuyo cumplimiento se consideró deficiente (P < 0,001). El análisis de la trayectoria asistencial reveló que en todos los contactos la tasa global de compleción con cumplimiento satisfactorio fue 61%.Conclusión: El TPI suministrado en el hogar a los niños que son contactos de un adulto con diagnóstico de TB y baciloscopia positiva alcanza altas tasas de aceptación y de cumplimiento que se pueden verificar. Los factores que determinan el éxito del TPI a escala nacional dependen del sistema de salud y no del paciente.

Optimising molecular diagnostic capacity for effective control of tuberculosis in high-burden settings.

The World Health Organization's 2035 vision is to reduce tuberculosis (TB) associated mortality by 95%. While low-burden, well-equipped industrialised economies can expect to see this goal achieved, it is challenging in the low- and middle-income countries that bear the highest burden of TB. Inadequate diagnosis leads to inappropriate treatment and poor clinical outcomes. The roll-out of the Xpert(®) MTB/RIF assay has demonstrated that molecular diagnostics can produce rapid diagnosis and treatment initiation. Strong molecular services are still limited to regional or national centres. The delay in implementation is due partly to resources, and partly to the suggestion that such techniques are too challenging for widespread implementation. We have successfully implemented a molecular tool for rapid monitoring of patient treatment response to anti-tuberculosis treatment in three high TB burden countries in Africa. We discuss here the challenges facing TB diagnosis and treatment monitoring, and draw from our experience in establishing molecular treatment monitoring platforms to provide practical insights into successful optimisation of molecular diagnostic capacity in resource-constrained, high TB burden settings. We recommend a holistic health system-wide approach for molecular diagnostic capacity development, addressing human resource training, institutional capacity development, streamlined procurement systems, and engagement with the public, policy makers and implementers of TB control programmes.

Corticotropin-releasing factor (CRF) agonists stimulate testosterone production in mouse leydig cells through CRF receptor-1.

The influence of CRF on testosterone production in primary mouse Leydig cell cultures was studied, and the type of CRF receptor (CRF-R) involved in this activity was determined. CRF directly stimulated testosterone production in mouse Leydig cells, but did not influence the maximum human (h)CG-induced testosterone production. The effect was time- and dose-dependent, saturable with an EC50 of 2.84 nM for hCRF, antagonized by the CRF antagonist alpha-helical CRF9-41, and accompanied by intracellular cAMP elevation. The rank order of potency of the natural CRF agonists, hCRF, ovine CRF, sauvagine, and urotensin, corresponded to that of their activities on CRF-R1 in rat pituitary cells and also to that reported for this receptor, but not for CRF-R2, when transfected into various cell lines. Furthermore, the difference in response of mouse Leydig cells to [11-D-Thr,12-D-Phe]- and [13-D-His,14-D-Leu]-ovine CRF corresponded to that measured when COS cells expressing CRF-R1 were activated, but was considerably smaller than that observed for activation of COS cells expressing CRF-R2alpha or -R2beta. The messenger RNA encoding the mouse CRF-R1 was detected by RT-PCR in mouse Leydig cell preparations. In contrast to mouse Leydig cells, CRF agonists had no influence on the basal testosterone and cAMP production by rat Leydig cells, nor did the agonists or antagonist change the hCG-stimulated testosterone and cAMP production by these cells. It is concluded that mouse Leydig cells express CRF-R1, mediating elevation of testosterone production by CRF agonists through cAMP. Because potencies of CRF agonists in activating mouse Leydig cells were more than 10-fold lower compared with their potencies in stimulating rat pituitary cells, it is suggested that the coupling of the CRF-R1 to intracellular signaling in Leydig cells is different from that in corticotropic pituitary cells, at least in quantitative terms.

Three-dimensional models of estrogen receptor ligand binding domain complexes, based on related crystal structures and mutational and structure-activity relationship data.

On the basis of the recently determined crystal structures of the ligand binding domains (LBDs) of the retinoic acid nuclear receptors (NRs), we present a three-dimensional (3D) molecular model of the human estrogen receptor alpha (hERalpha) LBD. A literature search for mutants affecting the binding properties has been performed; 45 out of 48 published mutants can be explained satisfactorily on the basis of the model. Estradiol has been docked into the binding pocket to probe its interactions with the protein. Energy minimizations and molecular dynamics calculations were performed for various ligand orientations. To evaluate their quality, the different models were scored using known structure-activity relationship (SAR) data for selected close estradiol homologues. The two best models explain largely the binding affinities of more distantly related ligands.

Gonadotropin-releasing hormone (GnRH) analogs: relationship between their structure, proteolytic inactivation and pharmacokinetics in rats.

There are two types of superactive agonists of gonadotropin-releasing hormone (GnRHa-I: (D-amino acid)6-GnRH and GnRHa-II: (D-amino acid)6-(desGly)10-GnRH- ethylamide) the high hormonal activity of which is understood to be due to their higher receptor affinity and their higher proteolytic stability as compared with the native GnRH sequence. Using the soluble fractions of various rat tissues in studies on the inactivation of GnRH peptides, we confirmed the higher proteolytic resistance of GnRHa-II, but not of D-Phe6-GnRH (GnRHa-I) and of another analog, D-Trp3-D-Phe6-GnRH, as compared with GnRH. The exact behaviour of the peptides during degradation was found to be dependent on the peptide concentrations used, showing the importance of using conditions as near to the physiological ones a possible. Towards the membrane fractions, however, the order of degradability was found to be GnRH much greater than D-Phe6-GnRH much greater than D-Trp3-D-Phe6-GnRH. The pharmacokinetic consequences of the different proteolytic degradabilities of the GnRH peptides, observed in rats, were a moderate increase in the biological half-life of D-Phe6-GnRH by 2.5-fold, as compared with GnRH, and a small increase in half-life of D-Trp3-D-Phe6-GnRH by 1.4-fold when compared with D-Phe6-GnRH. Whereas no intact GnRH was recovered in rat urine, small amounts of D-Phe6-GnRH (about 1% of dose) and high amounts of D-Trp3-D-Phe6-GnRH (25.5%) were excreted into urine. Combining the biochemical and pharmacokinetic data, it is concluded that proteolytic stability of GnRH analogs in pharmacological terms means stability towards membrane enzymes (pharmacologically-related stability) and that designing analogs with further increased proteolytic stability will be of only limited consequences with respect to their biological half-lives, the glomerular filtration rate of the kidney becoming the determining factor in the peptide clearance.

Gonadotropin-releasing hormone (GnRH) pharmacokinetics: peptide hormone pharmacokinetics needs clarification.

The plasma level curves of the peptide hormone gonadotropin-releasing hormone (GnRH) after its intravenous, intramuscular, and intraperitoneal administration into rats were fitted according to a two- (i.v.) and one-compartment model (i.m., i.p.), respectively. From the pharmacokinetic parameters it is concluded that urinary excretion and proteolytic degradation by kidney and liver are not sufficient to fully account for the clearance of the hormone and that, therefore, proteolytic degradation by tissues may play a role for the elimination of GnRH. This may be generally true with other short peptide hormones. The GnRH pharmacokinetics is shown as an example to underline that there presently exist problems of interpreting pharmacokinetic data of peptide hormones and that there is a need for a close interplay between biochemical and pharmacokinetic studies on peptide hormones for their pharmacokinetic behaviour to be understood.

Influence of continuous levels of fentanyl in rats on the mu-opioid receptor in the central nervous system.

The highly potent and efficacious mu-opioid agonist fentanyl was SC infused into rats with submaximal analgesic doses (0-1.14 mumol/kg/day) continuously for 8 days, checked by the constant daily urinary recovery of intact drug (0.43 +/- 0.031% of the daily dose). Tail-flick latencies measured at 24 (day 1) and 48 h (day 2) after starting the infusion were increased in a dose-dependent fashion compared with those before the infusion (day 0). However, at day 8, the latencies were increased only weakly, not significantly, revealing tolerance to the antinociceptive activity of fentanyl. Fentanyl at all doses showed no significant effect on the capacity (Bmax) and affinity (Kd) of the mu-opioid receptor binding of DAMGO to whole brain (Bmax 126.2 +/- 3.00 fmol/mg protein, Kd 1.00 +/- 0.04 nM) and spinal cord (Bmax 48.24 +/- 2.71 fmol/mg protein, Kd 1.93 +/- 0.13 nM) membranes gained from the rats after killing them at day 8. Gpp(NH)p increased the Kd for brain and spinal cord sites by 3.09 and 2.65, respectively, independent of the fentanyl dose. The infusion with fentanyl did not after the basal and forskolin-stimulated adenylate cyclase activity in the whole brain membranes, nor did it change the inhibition of the forskolin-stimulated activity by DAMGO. It is concluded that, in rats, constant long-term body levels of highly potent mu-agonists result in a tolerant state that, however, does not produce overall changes in the parameters of their specific receptor sites in the CNS, i.e., receptor capacity and affinity, and in the events closely related to them, i.e., their regulation by GTP and of adenylate cyclase. This does not exclude such possible changes to be restricted to specific regions in the CNS.

Increased pulmonary flow velocities in oversized homografts in patients after the Ross procedure.

OBJECTIVE: Between September 1991 and July 1996, 60 patients (mean age 29.8 +/- 9 years; range 5-57) underwent aortic root replacement with pulmonary autograft, a viable biologic and nondegenerating substitute. The pulmonary root was replaced with cryopreserved homografts from cardiac transplant recipients. The aim of this study was to evaluate differences in early valve function of viable and cryopreserved allografts. METHODS: All patients had Doppler echocardiographic examinations preoperatively, at discharge from hospital and 54 patients at 1 year follow-up. We measured aortic and pulmonary peak flow velocities with continuous and pulsed-wave Doppler, and graded aortic and pulmonary insufficiency (AI, PI) with color Doppler flow (grade 0-IV). Intraoperatively, the diameters of the pulmonary root and the pulmonary homograft were measured with standard valve probes and matched to body surface area. RESULTS: Pulmonary peak flow velocity (PVmax) increased significantly from preoperative 0.87 +/- 0.11 m/s to 1.30 +/- 0.34 m/s postoperatively (P < 0.001). The implanted homografts (mean 25.9 +/- 2.4 mm) were larger than their native pulmonary diameter (mean 23.3 +/- 1.8 mm) in all patients. Homograft size matched for body surface area (BSA) did not correlate with increased PVmax. There was a significant increase of PVmax at follow-up (FU) since discharge, also (1.83 +/- 0.53 m/s; P < 0.001). Pulsed-wave Doppler demonstrates that increase of PVmax is located directly at the homograft leaflets and not at the anastomoses. Aortic peak flow velocities (AVmax) were normal postoperatively and at FU (post = 1.35 +/- 0.35 m/s; FU = 1.17 +/- 0.27 m/s). There was no significant change in AI or PI since discharge (AI FU = 0.8 +/- 0.4; PI FU = 0.7 +/- 0.5). Eight patients with fever and symptoms diagnosed as post-pericardiotomy syndrome had significantly higher PVmax at FU (PVmax = 2.41 +/- 0.40 m/s; P < 0.02). CONCLUSIONS: The Ross procedure leads to normal AVmax but significant increase of PVmax even in oversized cryopreserved homografts immediately after surgery. Further increase of PVmax without changes in AVmax in the first year demonstrates that changes in flow velocities are valve related and not due to increase in cardiac output. Further investigations will be necessary to determine whether this observation is due to valve rejection or early leaflet degeneration and treatment with immunosuppressive therapy is warranted.

In-vivo release of a GnRH agonist from a slow-release poly(lactide-glycolide) copolymer preparation: comparison in rat, rabbit and guinea-pig.

Different batches of 50:50 poly((+-)-lactide-glycolide) copolymer (PLG) were used as biodegradable carriers for D-Phe6-gonadotropin-releasing hormone (GnRHa) in the form of injectable long-acting implants loaded with 10% GnRHa and tracer amounts of [125I]GnRHa. After their injection subcutaneously into rats, rabbits, and guinea-pigs, the release kinetics of the peptide were determined by counting the radioactivity remaining in the implants (i) after recovery from the rats after death or (ii) directly on the skin above the injection site of rabbits and guinea-pigs in-vivo. No significant differences in the release pattern of the peptide amongst the three species whether the release process was controlled by diffusion or by degradation of the polymeric matrix were found. It is concluded that the results of in-vivo release tests using laboratory animals are valid for man and that enzymes are not involved in the degradation of the polymeric matrix. The results may be of general importance for the use of long-term release PLG formulations of highly active drugs, especially peptides and proteins.

Disposition of 3H-labelled buserelin continuously infused into rats.

The disposition of the gonadotropin-releasing hormone (GnRH) agonist buserelin was studied in male rats under conditions of long-term administration. Rats were continuously infused with about 30 pmole [3H]-buserelin/24 h subcutaneously by osmotic minipumps for 4-7 days. After killing the rats, the 3H-activity of the tissues was measured and was found to be highly concentrated (about 10-fold to plasma) only in the pituitary. The daily amounts of 3H-activity excreted in urine and faces were constant over the whole infusion period, suggesting steady state conditions. On a molar basis, of the infused dose of buserelin, 14.8% was found to be excreted into urine as intact peptide, and 16.5, 10.8 and 20.6% as the partial buserelin sequences 1-2, 1-3 and 5-9. It is concluded that the major elimination route of buserelin, constant with time, is glomerular filtration, followed by enzymatic degradation of part of the filtered peptide by kidney tubuli enzymes to the partial sequences 1-2, 1-3 and 5-9, which reflects the proteolytic breakdown of buserelin by kidney membrane peptidases in vitro. Based on the similarities in the pharmacokinetics, in vivo metabolities, and in vitro enzymatic degradabilities among the GnRH agonists that have the native GnRH sequence modified at position 6 with or without additional modification at the C-terminal, the elimination process as shown here for buserelin should also be valid for other GnRH agonists.