Giulia Ghibellini

In vitro-in vivo correlation of hepatobiliary drug clearance in humans.

The biliary clearance (Clbiliary) of three compounds was estimated using sandwich‐cultured human hepatocytes (SCHH) and compared with Clbiliary values measured in vivo. Tc‐99m sestamibi (MIBI) Clbiliary was determined in seven healthy volunteers using an oroenteric catheter to aspirate duodenal secretions, and gamma scintigraphy to determine gallbladder contraction; this technique was used previously to determine Tc‐99m mebrofenin (MEB) and piperacillin (PIP) in vivo Clbiliary. In vitro Clbiliary of MEB, MIBI, and PIP was quantified in SCHH as the ratio of mass excreted into bile canaliculi and area under the blood concentration‐time curve (AUC) in medium. MIBI Clbiliary in vivo was 5.5±1.2 mL/min/kg (mean±SD). The rank order of Clbiliary predicted from SCHH corresponded well with the in vivo Clbiliary values in mL/min/kg for MEB (7.44 vs 16.1), MIBI (1.20 vs 5.51), and PIP (0.028 vs 0.032). In conclusion, the methods developed allowed for reproducible quantification of Clbiliary of drugs in healthy humans and prediction of Clbiliary from in vitro data.

A novel method for the determination of biliary clearance in humans

Biliary excretion is an important route of elimination and the biliary tract is a potential site of toxicity for many drugs and xenobiotics. Quantification of biliary excretion in healthy human volunteers is logistically challenging and is rarely defined during drug development. The current study uses a novel oroenteric tube coupled with a specialized clinical protocol to examine the pharmacokinetics of99mTechnetium (Tc-99m) mebrofenin, a compound that undergoes rapid hepatic uptake and extensive biliary excretion. A custommade multilumen oroenteric tube was positioned in the duodenum of healthy human volunteers. Subjects were positioned under a gamma camera and 2.5 mCi of Tc-99m mebrofenin was administered intravenously. Duodenal aspirates, blood samples, and urine were collected periodically for 3 hours. Two hours after Tc-99m mebrofenin administration, the gallbladder was contracted with an intravenous infusion of cholecystokinin-8. Gamma scintigraphy was used to determine the gallbladder ejection fraction in each subject. Total systemic clearance of Tc-99m mebrofenin approximated liver blood flow (Cltotal 17.3±1.7 mL/min/kg), and 35% to 84% of the Tc-99m mebrofenin dose was recovered in bile. However, when the data were corrected for the gallbladder ejection fraction, 71% to 92% of theexcreted Tc-99m mebrofenin dose was recovered. This novel croenteric tube and clinical protocol provide a useful method to quantify biliary excretion of xenobiotics in healthy human volunteers.

Effect of Ritonavir on 99mTechnetium–Mebrofenin Disposition in Humans: A Semi‐PBPK Modeling and In Vitro Approach to Predict Transporter‐Mediated DDIs

A semiphysiologically based pharmacokinetic (semi‐PBPK) model was developed to describe a unique blood, liver, and bile clinical data set for the hepatobiliary imaging agent 99mTechnetium–mebrofenin (99mTc–mebrofenin), and to simulate sites/mechanisms of a 99mTc–mebrofenin–ritonavir drug–drug interaction (DDI). The transport inhibitor ritonavir (multiple‐dose: 2 × 300 mg) significantly increased systemic 99mTc–mebrofenin exposure as compared with control (4,464 ± 1,861 vs. 1,970 ± 311 nCi min/ml; mean ± SD), without affecting overall hepatic exposure or biliary recovery. A novel extrahepatic distribution compartment was required to characterize 99mTc–mebrofenin disposition. Ritonavir inhibited 99mTc–mebrofenin accumulation in human sandwich‐cultured hepatocytes (SCH) (half maximal inhibitory concentration (IC50) = 3.46 ± 1.53 µmol/l). Despite ritonavir accumulation in hepatocytes, intracellular binding was extensive (97. 6%), which limited interactions with multidrug resistance protein 2 (MRP2)‐mediated biliary excretion. These in vitro data supported conclusions from modeling/simulation that ritonavir inhibited 99mTc–mebrofenin hepatic uptake, but not biliary excretion, at clinically relevant concentrations. This integrated approach, utilizing modeling, clinical, and in vitro data, emphasizes the importance of hepatic and extrahepatic distribution, assessment of inhibitory potential in relevant in vitro systems, and intracellular unbound concentrations to assess transporter‐mediated hepatic DDIs.

Neurodevelopmental attributes of joint hypermobility syndrome/Ehlers-Danlos syndrome, hypermobility type: Update and perspectives.

In the last decade, increasing attention has been devoted to the extra‐articular and extra‐cutaneous manifestations of joint hypermobility syndrome, also termed Ehlers–Danlos syndrome, hypermobility type (i.e., JHS/EDS‐HT). Despite the fact that the current diagnostic criteria for both disorders remain focused on joint hypermobility, musculoskeletal pain and skin changes, medical practice and research have started investigating a wide spectrum of visceral, neurological and developmental complications, which represent major burdens for affected individuals. In particular, children with generalized joint hypermobility often present with various neurodevelopmental issues and can be referred for neurological consultation. It is common that investigations in these patients yield negative or inconsistent results, eventually leading to the exclusion of any structural neurological or muscle disorder. In the context of specialized clinics for connective tissue disorders, a clear relationship between generalized joint hypermobility and a characteristic neurodevelopmental profile affecting coordination is emerging. The clinical features of these patients tend to overlap with those of developmental coordination disorder and can be associated with learning and other disabilities. Physical and psychological consequences of these additional difficulties add to the chief manifestations of the pre‐existing connective tissue disorder, affecting the well‐being and development of children and their families. In this review, particular attention is devoted to the nature of the link between joint hypermobility, coordination difficulties and neurodevelopmental issues in children. Presumed pathogenesis and management issues are explored in order to attract more attention on this association and nurture future clinical research.

In Vitro and In Vivo Determination of Piperacillin Metabolism in Humans

Piperacillin metabolism and biliary excretion are different between humans and preclinical species. In the present study, piperacillin metabolites were characterized in bile and urine of healthy humans and compared with metabolites formed in vitro. Volunteers were administered 2 g of piperacillin IV; blood, urine, and duodenal aspirates (obtained via a custom-made oroenteric catheter) were collected. The metabolism of piperacillin in humans also was investigated in vitro using pooled human liver microsomes and sandwich-cultured human hepatocytes. Piperacillin and metabolites were estimated by high-performance liquid chromatography with tandem mass spectrometry detection. Piperacillin, desethylpiperacillin, and desethylpiperacillin glucuronide were detected in bile, urine, and human liver microsomal incubates. Similar to the in vivo results, desethylpiperacillin was formed and excreted into bile canaliculi of sandwich-cultured human hepatocytes. This is the first report of glucuronidation of desethylpiperacillin in vitro or in vivo. The clinical method employed in this study to determine biliary clearance of drugs also facilitates bile collection as soon as bile is excreted from the gallbladder, thereby minimizing the exposure of labile metabolites to the intestinal environment. This study exemplifies how a combination of in vitro and in vivo tools can aid in the identification of metabolites unique to the human species.

Phenotypic variability in developmental coordination disorder: Clustering of generalized joint hypermobility with attention deficit/hyperactivity disorder, atypical swallowing and narrative difficulties

Developmental coordination disorder (DCD) is a recognized childhood disorder mostly characterized by motor coordination difficulties. Joint hypermobility syndrome, alternatively termed Ehlers–Danlos syndrome, hypermobility type (JHS/EDS‐HT), is a hereditary connective tissue disorder mainly featuring generalized joint hypermobility (gJHM), musculoskeletal pain, and minor skin features. Although these two conditions seem apparently unrelated, recent evidence highlights a high rate of motor and coordination findings in children with gJHM or JHS/EDS‐HT. Here, we investigated the prevalence of gJHM in 41 Italian children with DCD in order to check for the existence of recognizable phenotypic subgroups of DCD in relation to the presence/absence of gJHM. All patients were screened for Beighton score and a set of neuropsychological tests for motor competences (Movement Assessment Battery for Children and Visual‐Motor Integration tests), and language and learning difficulties (Linguistic Comprehension Test, Peabody Picture Vocabulary Test, Boston Naming Test, Bus Story Test, and Memoria‐Training tests). All patients were also screening for selected JHS/EDS‐HT‐associated features and swallowing problems. Nineteen (46%) children showed gJHM and 22 (54%) did not. Children with DCD and gJHM showed a significant excess of frequent falls (95 vs. 18%), easy bruising (74 vs. 0%), motor impersistence (89 vs. 23%), sore hands for writing (53 vs. 9%), attention deficit/hyperactivity disorder (89 vs. 36%), constipation (53 vs. 0%), arthralgias/myalgias (58 vs. 4%), narrative difficulties (74 vs. 32%), and atypical swallowing (74 vs. 18%). This study confirms the non‐causal association between DCD and gJHM, which, in turn, seems to increase the risk for non‐random additional features. The excess of language, learning, and swallowing difficulties in patients with DCD and gJHM suggests a wider effect of lax tissues in the development of the nervous system.

Assessment of biliary excretion in humans using a novel oroenteric tube

The biliary tract is an important route of elimination and potential site of toxicity for many xenobiotics. Quantification of biliary excretion in healthy volunteers is logistically challenging and is rarely defined during drug development. The current study uses a novel oroenteric tube to examine the pharmacokinetics of mebrofenin, a compound that undergoes rapid hepatic uptake and extensive biliary excretion. A custom‐made multi‐lumen oroenteric tube was positioned in the duodenum of four healthy volunteers (3 male, 1 female), subjects were positioned under a gamma camera and 2.5 mCi of 99mTc‐mebrofenin was administered intravenously. Duodenal aspirates, blood samples, and urine were collected periodically for 3 h. Sincalide (cholecystokinin‐8) was administered intravenously 2 h after mebrofenin to stimulate contraction of the gallbladder, and gamma scintigraphy was used to determine gallbladder ejection fraction. Total body clearance of mebrofenin approximated liver blood flow, and the oroenteric tube efficiently recovered the majority of excreted bile and mebrofenin. When corrected for gallbladder ejection fraction, 77–101% of the dose was recovered. This novel oroenteric tube and clinical protocol provide a useful method to quantify biliary excretion in healthy volunteers. (See Table )