In Vivo Methods to Study Uptake of Nanoparticles into the Brain

Several in vivo techniques have been developed to study and measure the uptake of CNS compounds into the brain. With these techniques, various parameters can be determined after drug administration, including the blood-to-brain influx constant (Kin), the permeability-surface area (PS) product, and the brain uptake index (BUI). These techniques have been mostly used for drugs that are expected to enter the brain via transmembrane diffusion or by carrier-mediated transcytosis. Drugs that have limitations in entering the brain via such pathways have been encapsulated in nanoparticles (based on lipids or synthetic polymers) to enhance brain uptake. Nanoparticles are different from CNS compounds in size, composition and uptake mechanisms. This has led to different methods and approaches to study brain uptake in vivo. Here we discuss the techniques generally used to measure nanoparticle uptake in addition to the techniques used for CNS compounds. Techniques include visualization methods, behavioral tests, and quantitative methods

Characterization of the resistance to the anorectic and endocrine effects of leptin in obesity-prone and obesity-resistant rats fed a high fat diet

Leptin produced by adipocytes controls body weight by restraining food intake and enhancing energy expenditure at the hypothalamic level. The diet-induced increase in fat mass is associated with the presence of elevated circulating leptin levels, suggesting the development of resistance to its anorectic effect. Rats, like humans, show different susceptibility to diet-induced obesity. The aim of the present study was to compare the degree of leptin resistance in obesity-prone (OP) vs obesity-resistant (OR) rats on a moderate high-fat (HF) diet and to establish if the effects of leptin on hypothalamo–pituitary endocrine functions were preserved. Starting from 6 weeks after birth, male Sprague–Dawley rats were fed on either a commercial HF diet (fat content: 20% of total calorie intake) or a standard pellet chow (CONT diet, fat content: 3%). After 12 weeks of diet, rats fed on HF diet were significantly heavier than rats fed on CONT diet. Animals fed on HF diet were ranked according to body weight; the two tails of the distribution were called OP and OR rats respectively. A polyethylene cannula was implanted into the right ventricle of rats 1 week before central leptin administration. After 12 weeks of HF feeding, both OR and OP rats were resistant to central leptin administration (10 μg, i.c.v.) (24 h calorie intake as a percent of vehicle-treated rats: CONT rats, 62 [50; 78]; OR, 93 [66; 118]; OP, 90 [70; 120] as medians and 95% confidence intervals (CIs) of six rats for each group). Conversely, after 32 weeks of diet both OR and OP rats were partially responsive to 10 μg leptin i.c.v. as compared with CONT rats (24 h calorie intake as a percent of vehicle-treated rats: CONT rats, 60 [50; 67]; OR, 65 [50; 80]; OP, 80 [60; 98] as medians and 95% CIs of six rats for each group); the decrease of food intake following 200 μg leptin i.p. administration was similar in all the three groups (calorie intake as a percent of vehicle-treated rats: 86 [80; 92] as median and 95% CI). The long-term intake of HF diet caused hyperleptinemia, hyperinsulinemia and higher plasma glucose levels in OP rats as compared with CONT rats. Plasma thyroxine (T4) was lower in all the rats fed the HF diet as compared with CONT. i.c.v. administration of leptin after 32 weeks of diet restored normal insulin levels in OP rats. Moreover, leptin increased plasma T4 concentration and strongly enhanced GH mRNA expression in the pituitary of OP as well as OR rats (18010% vs vehicle-treated rats). In conclusion, long-term intake of HF diet induced a partial central resistance to the anorectic effect of leptin in both lean and fat animals; the neuroendocrine effects of leptin on T4 and GH were preserved

Oral delivery of insulin loaded into peptide-conjugated polymeric nanoparticles in diabetic rats

The epidemic diffusion of overweight/obesity, together with physical inactivity, aging, urbanization, and population growth are the principal responsibles of the steadily increasing global prevalence of diabetes mellitus: from 2.8% in 2000 (= 171 million people) to a projected value of 4.4% in 2030 (= 366 million people). People with type 1 (insulin-dependent) diabetes mellitus, and also a consistent number of people with type 2 (non insulin-dependent) diabetes mellitus require multiple daily injections of insulin. It can be estimated that several dozens million people self-inject insulin every day. Thus, alternative routes of administration have been extensively investigated, especially the oral route. A successful oral formulation of insulin would have to bypass the two main barriers against the oral delivery of proteins: the enzymatic barrier of the gastrointestinal tract and the physical barrier made up of the intestinal epithelium. The most effective devices so far prepared have produced a maximum oral bioavailability of insulin of 13%, but a measurable absorption could be obtained only starting from the dose of 50 I.U.kg-1. We have recently demonstrated that polymeric nanoparticles (Np) made of the polyester poly(D,L-lactide-co-glycolide)(PLGA) conjugated with the simil-opioid glycosilated heptapeptide Gly-L-Phe-D-Th-Gly-L-Phe-L-Leu-L-Ser-(O-\u3b2-D-Glucose)-CONH2 (simil-opioid peptide-conjugated polymeric nanoparticles: SOP-Np) are able to cross the blood-brain barrier (BBB) much more effectively than the other so far prepared nanoparticles: the rationale of this approach lied on the known possibility for several opioid peptides to cross the BBB and other barriers, including the intestinal wall, by the activity of selective transport systems. Thus, aim of the present research was to investigate the suitability of SOP-Np as carriers for insulin across the intestinal barrier. Method: SOP-Np were prepared (185-220 nm size; polydispersity index 0.10-0.15; -35/-39 mV surface charge) and loaded with insulin (35 I.U./100 mg nanoparticles). Different amounts of such insulin-loaded SOP-Np were administered by oral gavage to diabetic rats, so to give 1, 3, or 10 I.U.kg-1 of insulin. A group of rats received by the same route 10 I.U.kg-1 of insulin in aqueous solution. Results: The aqueous solution of insulin produced only a negligible, non-significant decrease of glycemia; on the other hand, in rats treated with insulin-loaded SOP-Np, a dose-related increase of plasma insulin levels was observed, and a consequent, dose-related, and sustained decrease of glycemia was obtained: -50%, 90-180 min after the dose of 3 I.U.kg-1, -50/-70%, 120-240 min after the dose of 10 I.U.kg-1 (ANOVA followed by Bonferroni test: P<0.002/0.0001). Conclusion: The present results show that, in diabetic rats, nanoparticles of very low size, made of PLGA conjugated with a glycosilated simil-opioid heptapeptide, are able to effectively transport insulin across the intestinal barrier, preserving the biological activity of the hormone. If replicated in humans, these results could mean that the oral administration of insulin may be a concrete possibility

Participation of ATP-sensitive K+ channels in the peripheral antinociceptive effect of fentanyl in rats

We examined the effect of several K+ channel blockers such as glibenclamide, tolbutamide, charybdotoxin (ChTX), apamin, tetraethylammonium chloride (TEA), 4-aminopyridine (4-AP), and cesium on the ability of fentanyl, a clinically used selective µ-opioid receptor agonist, to promote peripheral antinociception. Antinociception was measured by the paw pressure test in male Wistar rats weighing 180-250 g (N = 5 animals per group). Carrageenan (250 µg/paw) decreased the threshold of responsiveness to noxious pressure (delta = 188.1 ± 5.3 g). This mechanical hyperalgesia was reduced by fentanyl (0.5, 1.5 and 3 µg/paw) in a peripherally mediated and dose-dependent fashion (17.3, 45.3 and 62.6%, respectively). The selective blockers of ATP-sensitive K+ channels glibenclamide (40, 80 and 160 µg/paw) and tolbutamide (80, 160 and 240 µg/paw) dose dependently antagonized the antinociception induced by fentanyl (1.5 µg/paw). In contrast, the effect of fentanyl was unaffected by the large conductance Ca2+-activated K+ channel blocker ChTX (2 µg/paw), the small conductance Ca2+-activated K+ channel blocker apamin (10 µg/paw), or the non-specific K+ channel blocker TEA (150 µg/paw), 4-AP (50 µg/paw), and cesium (250 µg/paw). These results extend previously reported data on the peripheral analgesic effect of morphine and fentanyl, suggesting for the first time that the peripheral µ-opioid receptor-mediated antinociceptive effect of fentanyl depends on activation of ATP-sensitive, but not other, K+ channels