Toward the establishment of standardized in vitro tests for lipid-based formulations. 5. Lipolysis of representative formulations by gastric lipase.

PURPOSE: Lipid-based formulations (LBF) are substrates for digestive lipases and digestion can significantly alter their properties and potential to support drug absorption. LBFs have been widely examined for their behaviour in the presence of pancreatic enzymes. Here, the impact of gastric lipase on the digestion of representative formulations from the Lipid Formulation Classification System has been investigated. METHODS: The pHstat technique was used to measure the lipolysis by recombinant dog gastric lipase (rDGL) of eight LBFs containing either medium (MC) or long (LC) chain triglycerides and a range of surfactants, at various pH values [1.5 to 7] representative of gastric and small intestine contents under both fasting and fed conditions. RESULTS: All LBFs were hydrolyzed by rDGL. The highest specific activities were measured at pH 4 with the type II and IIIA MC formulations that contained Tween®85 or Cremophor EL respectively. The maximum activity on LC formulations was recorded at pH 5 for the type IIIA-LC formulation. Direct measurement of LBF lipolysis using the pHstat, however, was limited by poor LC fatty acid ionization at low pH. CONCLUSIONS: Since gastric lipase initiates lipid digestion in the stomach, remains active in the intestine and acts on all representative LBFs, its implementation in future standardized in vitro assays may be beneficial. At this stage, however, routine use remains technically challenging.

Epidural neostigmine and clonidine improves the quality of combined spinal epidural analgesia in labour: a randomised, double-blind controlled trial.

BACKGROUND: In labour analgesia, the combination of epidural clonidine and neostigmine as adjuvants to local anaesthetics and opioids is under investigation to provide a longer duration of initial spinal analgesia with local anaesthetics and/or opioids. OBJECTIVES: To evaluate the quality of analgesia with epidural neostigmine and clonidine, added to initial spinal analgesia, and to test the hypothesis that the incidence of breakthrough pain could be reduced and patient satisfaction improved. DESIGN: Randomised double-blind controlled trial. SETTING: University Hospital of Leuven in Belgium. PARTICIPANTS: One hundred healthy, term (≥37 weeks) parturients. INTERVENTION: All patients received initial spinal analgesia with ropivacaine and sufentanil. Fifteen minutes after spinal injection, 10 ml of a solution containing neostigmine 500 µg and clonidine 75 µg, or 10 ml physiological saline alone was injected epidurally. Patient-controlled analgesia with ropivacaine and sufentanil was then made available. MAIN OUTCOME MEASURES: The incidence of breakthrough pain, patient satisfaction and hourly ropivacaine use. RESULTS: Ropivacaine use decreased significantly by 32.6% in the neostigmine/clonidine (NC) group [11.6 ±â€Š4.2 vs. 17.2 ±â€Š5.3 mg h in the NC group and placebo (P) group, respectively] and a significant difference in breakthrough pain was noted; only 3% in group NC had breakthrough pain compared with 36% in group P. Patient satisfaction was better after 1 h in group NC compared with group P (P <0.05) but not different after 24 h (visual analogue scale score 97 ±â€Š5 vs. 88 ±â€Š11 mm after 1 h; 92 ±â€Š10 vs. 90 ±â€Š14 mm after 24 h). CONCLUSION: The administration of epidural clonidine and neostigmine as adjuvants, following spinal injection of local anaesthetic, improves the quality of analgesia with less ropivacaine consumption, higher patient satisfaction 1 h after administration and a decrease in breakthrough pain compared to standard combined spinal and epidural analgesia and patient-controlled epidural analgesia with ropivacaine and sufentanil.

Toward the establishment of standardized in vitro tests for lipid-based formulations, part 3: understanding supersaturation versus precipitation potential during the in vitro digestion of type I, II, IIIA, IIIB and IV lipid-based formulations.

PURPOSE: Recent studies have shown that digestion of lipid-based formulations (LBFs) can stimulate both supersaturation and precipitation. The current study has evaluated the drug, formulation and dose-dependence of the supersaturation - precipitation balance for a range of LBFs. METHODS: Type I, II, IIIA/B LBFs containing medium-chain (MC) or long-chain (LC) lipids, and lipid-free Type IV LBF incorporating different doses of fenofibrate or tolfenamic acid were digested in vitro in a simulated intestinal medium. The degree of supersaturation was assessed through comparison of drug concentrations in aqueous digestion phases (APDIGEST) during LBF digestion and the equilibrium drug solubility in the same phases. RESULTS: Increasing fenofibrate or tolfenamic acid drug loads (i.e., dose) had negligible effects on LC LBF performance during digestion, but promoted drug crystallization (confirmed by XRPD) from MC and Type IV LBF. Drug crystallization was only evident in instances when the calculated maximum supersaturation ratio (SR(M)) was >3. This threshold SR(M) value was remarkably consistent across all LBF and was also consistent with previous studies with danazol. CONCLUSIONS: The maximum supersaturation ratio (SR(M)) provides an indication of the supersaturation 'pressure' exerted by formulation digestion and is strongly predictive of the likelihood of drug precipitation in vitro. This may also prove effective in discriminating the in vivo performance of LBFs.

Toward the establishment of standardized in vitro tests for lipid-based formulations. 2. The effect of bile salt concentration and drug loading on the performance of type I, II, IIIA, IIIB, and IV formulations during in vitro digestion.

The LFCS Consortium was established to develop standardized in vitro tests for lipid-based formulations (LBFs) and to examine the utility of these tests to probe the fundamental mechanisms that underlie LBF performance. In this publication, the impact of bile salt (sodium taurodeoxycholate, NaTDC) concentration and drug loading on the ability of a range of representative LBFs to generate and sustain drug solubilization and supersaturation during in vitro digestion testing has been explored and a common driver of the potential for drug precipitation identified. Danazol was used as a model poorly water-soluble drug throughout. In general, increasing NaTDC concentrations increased the digestion of the most lipophilic LBFs and promoted lipid (and drug) trafficking from poorly dispersed oil phases to the aqueous colloidal phase (AP(DIGEST)). High NaTDC concentrations showed some capacity to reduce drug precipitation, although, at NaTDC concentrations ≥3 mM, NaTDC effects on either digestion or drug solubilization were modest. In contrast, increasing drug load had a marked impact on drug solubilization. For LBFs containing long-chain lipids, drug precipitation was limited even at drug loads approaching saturation in the formulation and concentrations of solubilized drug in AP(DIGEST) increased with increased drug load. For LBFs containing medium-chain lipids, however, significant precipitation was evident, especially at higher drug loads. Across all formulations a remarkably consistent trend emerged such that the likelihood of precipitation was almost entirely dependent on the maximum supersaturation ratio (SR(M)) attained on initiation of digestion. SR(M) defines the supersaturation "pressure" in the system and is calculated from the maximum attainable concentration in the AP(DIGEST) (assuming zero precipitation), divided by the solubility of the drug in the colloidal phases formed post digestion. For LBFs where phase separation of oil phases did not occur, a threshold value for SR(M) was evident, regardless of formulation composition and drug solubilization reduced markedly above SR(M) > 2.5. The threshold SR(M) may prove to be an effective tool in discriminating between LBFs based on performance.

Impact of Vitamin E supplementation on lipoprotein peroxidation and composition in Type 1 diabetic patients treated with Atorvastatin.

OBJECTIVE: To investigate the impact of Vitamin E on lipids and peroxidation during statin treatment. RESEARCH DESIGN AND METHODS: T1DM patients with high cholesterol received Atorvastatin 20mg with either placebo (group AP, n = 11) or d-alpha-tocopherol 750 IU (group AE, n = 11) daily. They were monitored for blood biochemistry, low-density lipoprotein (LDL) subfractions and lipid peroxidation at inclusion and after 3 and 6 months. RESULTS: Serum cholesterol and triglycerides decreased to the same extent (29 and 21% respectively) in both groups. Serum tocopherol decreased by 18% in AP and increased by 50% in AE (P < 0.0001, between-group comparison by repeated measures ANOVA) but relative to lipids it increased by 15% in AP and by 100% in AE. Copper-induced production of thiobarbituric reactive substances in the LDL + VLDL fraction increased by 18% in AP and did not change in AE (P = 0.02). The lagtime for the production of fluorescent products was prolonged by 13 min only in group AE (P = 0.028). Plasma malondialdehyde decreased by 35% in both groups (P = 0.002) but not when adjusted for lipids. CONCLUSIONS: In T1DM Vitamin E supplements do not affect the lowering of lipids and plasma malondialdehyde achieved by Atorvastatin. They reverse the increase of in vitro peroxidation caused by Atorvastatin but do not achieve the decreases observed in patients not receiving lipid-lowering drugs. These results indicate that the antioxidant effect of Vitamin E is attenuated when given in conjunction with this statin.

Evolution of serum alpha-tocopherol in the postprandial and postabsorptive phases in type 1 diabetes mellitus.

Blood glucose, lipids, alpha-tocopherol, and malondialdehyde were monitored in type 1 diabetes mellitus patients for 8 hours after a standard fat-rich breakfast and lunch. Although glucose and triglycerides increased, alpha-tocopherol decreased and malondialdehyde increased in the postprandial phase. In the postabsorptive phase values returned to fasting levels. These results point to the possible relevance of postprandial alpha-tocopherol depletion and lipid peroxidation to an increased cardiovascular risk in these patients.

Toward the establishment of standardized in vitro tests for lipid-based formulations, part 4: proposing a new lipid formulation performance classification system.

The Lipid Formulation Classification System Consortium looks to develop standardized in vitro tests and to generate much-needed performance criteria for lipid-based formulations (LBFs). This article highlights the value of performing a second, more stressful digestion test to identify LBFs near a performance threshold and to facilitate lead formulation selection in instances where several LBF prototypes perform adequately under standard digestion conditions (but where further discrimination is necessary). Stressed digestion tests can be designed based on an understanding of the factors that affect LBF performance, including the degree of supersaturation generated on dispersion/digestion. Stresses evaluated included decreasing LBF concentration (↓LBF), increasing bile salt, and decreasing pH. Their capacity to stress LBFs was dependent on LBF composition and drug type: ↓LBF was a stressor to medium-chain glyceride-rich LBFs, but not more hydrophilic surfactant-rich LBFs, whereas decreasing pH stressed tolfenamic acid LBFs, but not fenofibrate LBFs. Lastly, a new Performance Classification System, that is, LBF composition independent, is proposed to promote standardized LBF comparisons, encourage robust LBF development, and facilitate dialogue with the regulatory authorities. This classification system is based on the concept that performance evaluations across three in vitro tests, designed to subject a LBF to progressively more challenging conditions, will enable effective LBF discrimination and performance grading.

Toward the establishment of standardized in vitro tests for lipid-based formulations, part 6: effects of varying pancreatin and calcium levels.

The impact of pancreatin and calcium addition on a wide array of lipid-based formulations (LBFs) during in vitro lipolysis, with regard to digestion rates and distribution of the model drug danazol, was investigated. Pancreatin primarily affected the extent of digestion, leaving drug distribution somewhat unaffected. Calcium only affected the extent of digestion slightly but had a major influence on drug distribution, with more drug precipitating at higher calcium levels. This is likely to be caused by a combination of removal of lipolysis products from solution by the formation of calcium soaps and calcium precipitating with bile acids, events known to reduce the solubilizing capacity of LBFs dispersed in biorelevant media. Further, during the digestion of hydrophilic LBFs, like IIIA-LC, the un-ionized-ionized ratio of free fatty acids (FFA) remained unchanged at physiological calcium levels. This makes the titration curves at pH 6.5 representable for digestion. However, caution should be taken when interpreting lipolysis curves of lipophilic LBFs, like I-LC, at pH 6.5, at physiological levels of calcium (1.4 mM); un-ionized-ionized ratio of FFA might change during digestion, rendering the lipolysis curve at pH 6.5 non-representable for the total digestion. The ratio of un-ionized-ionized FFAs can be maintained during digestion by applying non-physiological levels of calcium, resulting in a modified drug distribution with increased drug precipitation. However, as the main objective of the in vitro digestion model is to evaluate drug distribution, which is believed to have an impact on bioavailability in vivo, a physiological level (1.4 mM) of calcium is preferred.

Toward the establishment of standardized in vitro tests for lipid-based formulations, part 1: method parameterization and comparison of in vitro digestion profiles across a range of representative formulations.

The Lipid Formulation Classification System Consortium is an industry-academia collaboration, established to develop standardized in vitro methods for the assessment of lipid-based formulations (LBFs). In this first publication, baseline conditions for the conduct of digestion tests are suggested and a series of eight model LBFs are described to probe test performance across different formulation types. Digestion experiments were performed in vitro using a pH-stat apparatus and danazol employed as a model poorly water-soluble drug. LBF digestion (rate and extent) and drug solubilization patterns on digestion were examined. To evaluate cross-site reproducibility, experiments were conducted at two sites and highly consistent results were obtained. In a further refinement, bench-top centrifugation was explored as a higher throughput approach to separation of the products of digestion (and compared with ultracentrifugation), and conditions under which this method was acceptable were defined. Drug solubilization was highly dependent on LBF composition, but poorly correlated with simple performance indicators such as dispersion efficiency, confirming the utility of the digestion model as a means of formulation differentiation.

Minimally-invasive and non-invasive continuous glucose monitoring systems: indications, advantages, limitations and clinical aspects.

Accurate and reliable devices sensing glucose on a (near)-continuous basis may facilitate specific therapeutic adjustments that need to be made to avoid hypo- and hyperglycaemic excursions, thereby improving metabolic control. Current continuous glucose monitoring (CGM) systems indicate the glucose level, the direction and magnitude of change of glucose levels, and can be used to assess glycaemic variability. In addition, real-time CGM sensors can serve as a tool to predict impending glucose excursions, thereby providing alarm signals of hypo- and hyperglycaemic values warning the patient to take preventative actions. Quality of life may also improve by using CGM via reducing the fear of hypoglycaemia. Particularly patients with brittle diabetes, hypoglycaemia unawareness, gastroparesis, pregnant women, or pump users, who are motivated to participate in their diabetes care and are technologically adept, may benefit from CGM. However, to successfully implement CGM in daily practice, these devices must be accurate and reliable, and one must be aware of the limitations of current CGM systems, that originate from physiological and technical aspects. Whether CGM succeeds in improving metabolic control, reducing hypoglycaemic episodes, and improving quality of life in the majority of patients remains to be proven. Should this be the case, real-time CGM may reduce chronic diabetic complications, and avoid hospitalisations, thereby reducing health care costs. In this article we will review indications, advantages, limitations, clinical and technical aspects of current minimally-invasive and non-invasive CGM sensors.

Time course of oxidative stress status in the postprandial and postabsorptive states in type 1 diabetes mellitus: relationship to glucose and lipid changes.

OBJECTIVE: The aim of this study was to compare oxidative stress status (OSS) with blood glucose and lipid changes during the fasting, postprandial and postabsorptive phases in type 1 diabetes mellitus. METHODS: Twenty-three patients on intensive insulin treatment received a standard fat-rich breakfast and lunch. OSS was monitored at fasting (F), just after the post-breakfast glycemia peak (BP) (identified by continuous subcutaneous glucose monitoring), 3.5-h post-breakfast (B3.5), just after the post-lunch peak (LP), just after the post-lunch dale (LD) and 5 hours after lunch (L5). RESULTS: Whereas whole blood glutathione and plasma protein thiols increased in the postprandial period (from 6.52 +/- 1.20 (F) to 7.08 +/- 1.45 micromol/g Hb (BP), p = 0.005), ascorbate decreased gradually from 44 +/- 17 (F) to 39 +/- 19 micromol/L (LD), p = 0.015. Retinol and alpha-tocopherol also decreased from 27.1 +/- 7.0 (F) to 25.3 +/- 5.2 micromol/L (BP), p = 0.005. Uric acid decreased later, from 213 +/- 77 (BP) to 204 +/- 68 micromol/L (B3.5), p = 0.01, but then increased in LP (231 +/- 70 micromol/L) and LD to values higher than F (215 +/- 64, micromol/L, p = 0.01). Malondialdehyde increased gradually from 1.02 +/- 0.36 (F) to a maximum of 1.14 +/- 0.40 micromol/L (LP). In the postabsorptive phase (L5) all parameters except for thiols reverted to fasting concentrations. CONCLUSIONS: In type 1 diabetes lipid peroxidation increases during the postprandial phase in parallel to glucose and triglyceride changes. Blood antioxidants, however, followed diverse patterns of change.

Effects of Atorvastatin on LDL sub-fractions and peroxidation in type 1 diabetic patients: a randomised double-blind placebo-controlled study.

BACKGROUND: Patients with diabetes have an increased risk of both developing and dying from cardiovascular disease, and, currently, more aggressive lipid-lowering targets are being recommended for these patients. Statins are widely and successfully used to correct dyslipidemia and prevent acute coronary episodes, but their effects on lipoprotein composition and peroxidation have not been fully investigated. We aimed to address this issue in type 1 diabetes mellitus. METHODS: T1DM patients with atherogenic index (total/HDL-cholesterol > 4) were randomised double-blindly to group A (n = 12) that received Atorvastatin 40 mg/day and group P (n = 12) that received placebo. They were monitored for blood biochemistry, LDL sub-fractions and lipid peroxidation at inclusion, after 6 and after 12 weeks. RESULTS: In group A, the 40% decrease in serum total and LDL cholesterol and 20% decrease in triglycerides was accompanied by a decrease in serum alpha-tocopherol from 46.4 +/- 16.3 (mean +/- SD) at inclusion to 32.2 +/- 11.8 and 32.6 +/- 14.0 micromol/L after 6 and 12 weeks respectively (p < 0.001 compared to group P by repeated-measures ANOVA). Relative to LDL + VLDL cholesterol, alpha-tocopherol increased by 40% (p < 0.001). Copper-induced LDL + VLDL peroxidation increased from 4891 +/- 1325 at inclusion to 6821 +/- 2291 and 7040 +/- 1712 nmol TBARS/mg LDL + VLDL cholesterol produced in 3 h (p = 0.004). LDL sub-fractions shifted towards the less dense regions (p = 0.03). CONCLUSIONS: These results suggest that Atorvastatin lowers the antioxidant capacity of LDL and VLDL in T1DM. The mechanisms underlying these changes merit further investigation and should be taken into account when planning long-term primary prevention of CHD in diabetes.

Postprandial glucose monitoring in type 1 diabetes mellitus: use of a continuous subcutaneous monitoring device.

BACKGROUND: Pre-prandial glucose gives insufficient information on glycemic excursions throughout the day. We aimed to test a continuous subcutaneous glucose-monitoring device (GlucoDay) to describe postprandial glucose changes. METHODS: In 23 T1DM patients, 24-h GlucoDay registrations were started about 14 h before receiving a standard breakfast B and 3 h later lunch L. RESULTS: The 3-min glucose values were computed into parameters describing the postprandial changes after B and L. Two-hour glucose was higher after B (243 +/- 69 vs 180 +/- 79 mg/dL after L, p < 0.0001). Maximum glycemia (313 +/- 105 mg/dL after B and 304 +/- 119 after L, p < 0.0001) was higher and was reached after 78 and 57 min respectively. Three-hour AUC was higher but 30-min AUC was lower after B (5725 +/- 2414 vs 7488 +/- 2208 min mg/dL after L, p = 0.004). Glucose spikes (maximum peak minus fasting plasma glucose) were similar after B and L but the difference between maximum and minimum values was smaller after B (165 +/- 110 vs 219 +/- 115 mg/dL after L, p = 0.020). Duration of hyperglycemic periods >200, 140 or 126 mg/dL were not different after B or L, but time spent at glucose <100 mg/dL was longer after L (p < 0.0001). CONCLUSIONS: These results illustrate the use of subcutaneous glucose registration to characterize postprandial glycemia patterns in T1DM. Application of such methods to evaluate this and other clinical situations in DM can lead to therapeutic and dietary adjustments and ultimately improve glycemic control.