In vitro and in vivo immunogenicity assessment of protein aggregate characteristics.

The immunogenicity risk of therapeutic protein aggregates has been extensively investigated over the past decades. While it is established that not all aggregates are equally immunogenic, the specific aggregate characteristics, which are most likely to induce an immune response, remain ambiguous. The aim of this study was to perform comprehensive in vitro and in vivo immunogenicity assessment of human insulin aggregates varying in size, structure and chemical modifications, while keeping other morphological characteristics constant. We found that flexible aggregates with highly altered secondary structure were most immunogenic in all setups, while compact aggregates with native-like structure were found to be immunogenic primarily in vivo. Moreover, sub-visible (1-100 µm) aggregates were found to be more immunogenic than sub-micron (0.1-1 µm) aggregates, while chemical modifications (deamidation, ethylation and covalent dimers) were not found to have any measurable impact on immunogenicity. The findings highlight the importance of utilizing aggregates varying in few characteristics for assessment of immunogenicity risk of specific morphological features and may provide a workflow for reliable particle analysis in biotherapeutics.

Metabolism of peptide YY 3-36 in Göttingen mini-pig and rhesus monkey.

Peptide YY 3-36-amide (PYY3-36) is a peptide hormone, which is known to decrease appetite and food-intake by activation of the Y2 receptor. The current studies were designed to identify the metabolites of PYY3-36 in mini-pig and rhesus monkey. Plasma samples were analyzed by high resolution LC-MS (and MS/MS) in order to unambiguously identify the metabolites of PYY3-36. In summary, the metabolism of PYY3-36 was similar in mini-pig and rhesus monkey. Several metabolites were identified and PYY3-34 was identified at the highest levels in plasma. In addition, mini-pigs were also dosed with PYY1-36-amide, PYY3-35, PYY3-34 and [N-methyl 34Q]-PYY3-36-amide in order to investigate the mechanisms by which PYY was metabolized. PYY3-35 was rapidly converted to PYY3-34 whereas dosing of PYY3-34 to mini-pigs only showed circulating degradation products at low levels, i.e., PYY3-34 was metabolically more stable than PYY3-36 and PYY3-35. [N-methyl 34Q]-PYY3-36-amide was hypothesized to be stable toward cleavage between 34Q and 35R and after i.v. administration to mini-pigs, one major cleavage product was identified as [N-methyl 34Q]-PYY3-35. Overall, this showed that cleavage between 35R and 36Y was possible as well as between 34Q and 35R (as shown for PYY3-35), which indicated that metabolism of PYY3-36 to PYY3-34 may be a two-step process. PYY1-36 was also dosed to mini-pigs, which showed that PYY1-36 was metabolized in the C-terminal as PYY3-36. The overall degradation pattern of PYY1-36 was more complex due to the simultaneous enzymatic degradation in the N-terminal to form PYY2-34/36 and PYY3-34/36. In vitro incubations with heparin stabilized plasma showed that PYY3-36 was degraded with a half-life of 175 min, whereas incubations with PYY3-35 (half-life of 6 min) showed a rapid formation of PYY3-34. In conclusion, the present studies showed that PYY3-36 underwent enzymatic degradation in the C-terminal part and that the major circulating metabolite was PYY3-34. Furthermore, it may be a sequential two-step process leading to the formation of PYY3-35 and subsequently the metabolically more stable PYY3-34.

Predictors of mortality in patients with type 2 diabetes with or without diabetic nephropathy: a follow-up study.

OBJECTIVE: To evaluate the prognostic significance of cardiovascular risk factors including 24-h ambulatory blood pressure level and rhythm for all-cause mortality in type 2 diabetic patients. METHODS: In a prospective observational study, 104 patients with type 2 diabetes were followed: 51 patients with diabetic nephropathy and 53 patients with persistent normoalbuminuria. At baseline, 24-h ambulatory blood pressure, left ventricular hypertrophy, glomerular filtration rate and cardiac autonomic neuropathy were measured. Blood samples were taken and patients answered a World Health Organization questionnaire. Dipping was calculated as the average nocturnal reduction in systolic and diastolic blood pressure. RESULTS: Mean follow-up was 9.2 years (range 0.5-12.9). During follow-up, 54 of 104 patients died. Sixteen patients (15%) had higher blood pressure at night than during the day (reversed pattern); 14 of these patients died (88%), compared to 40 of 88 patients (45%) with reduced dipping or normal dipping; log rank P = 0.001. In a Cox regression analysis, predictors of all-cause mortality were: age, male sex, presence of left ventricular hypertrophy, glycated haemoglobin A1c (HbA1c), daytime systolic blood pressure, cardiac autonomic neuropathy, glomerular filtration rate and dipping (1% increase; hazard ratio 0.97, 95% confidence interval 0.94-0.998, P = 0.033). CONCLUSION: Type 2 diabetes patients with non-dipping of night blood pressure were at higher risk of death as compared to dippers, independent of known cardiovascular risk factors. Since non-dipping has a high prevalence in patients with diabetic nephropathy, 24-h ambulatory blood pressure should be used to assess a full risk profile and blood pressure-lowering therapy in these patients.

Characterization of prototype self-nanoemulsifying formulations of lipophilic compounds.

This study describes the evaluation and characterization of a self-nanoemulsifying drug delivery system (SNEDDS) consisting of a nonionic surfactant (Cremophor RH40), a mixture of long chain mono-, di-, and triacylglycerides (Maisine 35-1 and Sesame oil) and ethanol. Compositions containing 10% (w/w) ethanol, 40%-60% (w/w) lipid content, and 30%-50% (w/w) Cremophor RH40 were identified as pharmaceutically relevant, robust, and self-nanoemulsifying when dispersed in aqueous media. The influence of adding three different lipophilic model drug compounds (danazol, halofantrine, and probucol) to the SNEDDS was evaluated. While danazol precipitated from the SNEDDS after dispersion in aqueous media, halofantrine and procubol remained solubilized. Halofantrine- and procubol-loaded SNEDDS were evaluated in both saline and in media simulating fasted and fed-state intestinal fluid (FaSSIF and FeSSIF) using dynamic light scattering and small-angle X-ray scattering (SAXS) techniques. Stable nanoemulsions with droplet sizes in the range of 20-50 nm were formed in all media and with and without drugs. The mean size of the droplets was neither affected significantly by being dispersed into the media simulating gastro intestinal fluid, nor by addition of the drug.

Clinical studies with oral lipid based formulations of poorly soluble compounds.

This work is an attempt to give an overview of the clinical data available on lipid based formulations. Lipid and surfactant based formulations are recognized as a feasible approach to improve bioavailability of poorly soluble compounds. However not many clinical studies have been published so far. Several drug products intended for oral administration have been marketed utilizing lipid and surfactant based formulations. Sandimmune((R)) and Sandimmune Neoral((R)) (cyclosporin A, Novartis), Norvir((R)) (ritonavir), and Fortovase((R)) (saquinavir) have been formulated in self-emulsifying drug delivery systems (SEDDS). This review summarizes published pharmacokinetic studies of orally administered lipid based formulations of poorly aqueous soluble drugs in human subjects. Special attention has been paid to the physicochemical characteristics of the formulations, when available and the impact of these properties on the in vivo performance of the formulation. Equally important is the effect of concurrent food intake on the bioavailability of poorly soluble compounds. The effect of food on the bioavailability of compounds formulated in lipid and surfactant based formulations is also reviewed.

Myocardial perfusion in type 2 diabetes with left ventricular hypertrophy: normalisation by acute angiotensin-converting enzyme inhibition.

The purpose of this study was to assess whether acute angiotensin-converting enzyme (ACE) inhibition would improve myocardial perfusion and perfusion reserve in a subpopulation of normotensive patients with diabetes and left ventricular hypertrophy (LVH), both independent risk factors of coronary disease. Using positron emission tomography (PET), we investigated the response of regional myocardial perfusion to acute ACE inhibition with i.v. infusion of perindoprilat (vs saline infusion as control, minimum interval 3 days) in 12 diabetic patients with LVH. Myocardial perfusion was quantified with PET using nitrogen-13 ammonia infused at rest and during dipyridamole hyperaemia. Twelve healthy control subjects were included in the study, five of whom were also studied with perindoprilat. Mean blood pressure in normo-albuminuric, asymptomatic patients was 123+/-7/65+/-9 mmHg. Compared with controls, maximal perfusion was reduced in patients (1.8+/-0.6 vs 2.5+/-1.0 ml min(-1) g(-1); P<0.05), and perfusion reserve was also lower, at borderline significance (2.7+/-1.0 vs 3.6+/-1.3; P=0.059). During perindoprilat infusion, myocardial perfusion reserve in patients increased to 3.9+/-0.9 ( P<0.001) due to normalisation of maximal perfusion (2.3+/-0.5 ml min(-1) g(-1), P<0.01). In the five control subjects both resting and hyperaemic perfusion remained unchanged during perindoprilat infusion. It is concluded that acute ACE inhibition with perindoprilat improves maximal achieved myocardial perfusion in non-hypertensive patients with diabetes and LVH.

Dipyridamole, cold pressor test, and demonstration of endothelial dysfunction: a PET study of myocardial perfusion in diabetes.

UNLABELLED: Much evidence suggests endothelial dysfunction to be present in non-insulin-dependent diabetes mellitus (NIDDM) and to be important for the development of myocardial ischemia. Endothelial function in the coronary vessels may be studied in various ways. We compared the effect of cold pressor testing (CPT) with that of dipyridamole, a pharmacologic vasodilator, on coronary blood flow (CBF) measured by PET in NIDDM patients and healthy volunteers. In addition, we studied the effect of acute angiotensin-converting enzyme (ACE) inhibition on the flow response. METHODS: Ten NIDDM patients and 10 control subjects participated. Myocardial perfusion was determined at baseline, during CPT, and after dipyridamole infusion by PET using intravenous (13)N-ammonia. RESULTS: Resting CBF was similar in NIDDM patients and in control subjects. CPT increased CBF by 20% in the control group, whereas no increase was observed in the patients. After dipyridamole infusion, CBF increased 2- to 3-fold in patients and 3- to 4-fold in control subjects. The increase and maximal CBF were significantly higher in control subjects than in patients. During ACE-inhibitor infusion, which had no influence on resting CBF in patients or control subjects (n = 5), CPT increased CBF by 14% in the NIDDM group. After dipyridamole, CBF increased 3- to 4-fold in both groups. The increase in CBF and maximal CBF in the 2 groups were not different during ACE-inhibitor infusion. CONCLUSION: In these NIDDM patients without evidence of epicardial coronary disease, endothelial dysfunction is strongly suggested by an impaired increase in CBF both to dipyridamole and to CPT. This dysfunction was reversed by infusion of an ACE inhibitor. Although ACE inhibition during CPT did induce significant increases in CBF in the patients, the changes during ACE inhibition were small compared with the dipyridamole response, and the absence of CBF increase during CPT in 3 of the 10 control subjects further limits the value of CPT for the study of coronary endothelial dysfunction.