Enhanced in vivo absorption of CB-1 antagonist in rats via solid solutions prepared by hot-melt extrusion.

The aim of the present work was to investigate in vitro dissolution properties of three binary solid solutions prepared by a hot-melt extrusion (HME) process with vinyl pirrolidone--vinyl acetate copolymer (Kollidon VA 64), ethyl acrylate, methyl methacrylate polymer (Eudragit E) polyetilenglicol 8000 (PEG 8000) with a cannabinoid type 1 (CB-1) antagonist. Hansen solubility parameters were calculated from the chemical structures of the drug and the individual polymers in order to predict miscibility. Solid state characterizations of drug substance, physical blends and HME formulations were performed with differential scanning calorimetry. The dissolution testing conducted under sink conditions revealed that the dissolution rate of HME formulations improved around 1.8-fold vs drug substance. Supersaturation dissolution study demonstrated that HME formulations composed by Eudragit E and Kollidon VA64 increased drug solubility between 30- and 35-fold, respectively comparing to the drug substance. Physical and chemical stability of formulations were studied at 40°C/75%HR with open dish during 15 days. The formulation composed by the drug and Eudragit E at 10:90 was evaluated for in vivo drug absorption in male Wistar-Hannover rats and it was found to increase CB-1 absorption threefold greater than pure drug oral suspension.

Simultaneous technetium-99 MAG(3), iodine-131-orthoiodohippurate and iodine-125 iothalamate clearance and biodistribution after bolus injection in rats.

A bolus injection multiple blood sampling method was developed for the simultaneous measurement of blood and plasma clearance of three radiopharmaceuticals in rats. Technetium-99m mercaptoacetyltriglycine ([(99m)Tc]MAG(3)) and iodine-131-orthoiodohippurate ([(131)I]OIH) were used as makers of effective renal blood flow (ERBF), and iodine-125 iothalamate ([(125)I]IOT) was used as a marker of glomerular filtration rate (GFR). These methods can be easily performed in rats without arterial catheterization. Tissue biodistribution was studied in four groups of rats subjected to the following: group A, renal pedicle isolation (sham-operated); group B, ligature of one kidney pedicle; group C, ligature of both renal pedicles; and group D, ligature of both kidney pedicles and the bile duct. Renal clearance of [(99m)Tc]MAG(3) was greater than [(131)I]OIH and both agents were cleared faster than ([(125)I]-IOT). Either of the two markers of ERBF may be used in experimental studies, but it should be borne in mind that these are relative measurements of kidney performance. [(99m)Tc]MAG(3) and [(125)I]-IOT showed bile excretion in healthy rats, so they cannot completely fulfill the requirements for use as markers of ERBF. When renal function was impaired experimentally, [(99m)Tc]MAG(3) and [(125)I]-IOT were excreted in bile and [(131)I]OIH was secreted in the intestine. Thus, while the markers of ERBF and GFR may be reliable under normal physiological conditions, they may give progressively more erroneous values as renal function deteriorates.

Characterization of allergoids from ovalbumin in vitro and in vivo.

Several in vivo and in vitro methods for monitoring immunological properties of two allergoids obtained by formaldehyde treatment of ovalbumin (OA) were developed. The calculated molecular weight of allergoids was 80 kD (OA-F1) and 165 kD (OA-F2), respectively. The allergenic activity in vitro of allergoids in mast-cell histamine release assay was 1000 times lower than of OA. Both allergoids showed reduced ability to induce passive cutaneous anaphylaxis in the Sprague-Dawley rats or systemic anaphylaxis in Dunkin-Harley guinea-pigs. The ability of OA and allergoids to bind to the OA-specific IgE antibodies was measured in vivo by the inhibition of passive cutaneous anaphylaxis (PCA-inhibition). Allergoid binding to IgE was 51-66% lower than the native allergen. Moreover, the avidity of OA-specific IgG antibodies, measured by ELISA-inhibition, for allergoids and allergen was of the same order. Allergoids induced a different pattern of humoral immune response from that, induced by the native allergen. Thus, after immunization of BALB/c mouse, both allergoids induced a higher production of IgG and a lower production of IgE than OA, only OA-F2 induced a lower production of IgG1. The differences in the IgA response to the immunogens was not significant. Delayed hypersensitivity studies in the BALB/c mouse showed that allergoids were 5- to 12-times less effective in inducing a cell-mediated immune response than OA. The present study provides a battery of immunological methods for preclinical testing of modified allergens.

Age-dependent enzymuria, proteinuria and changes in renal blood flow and glomerular filtration rate in rats.

The time course of urinary excretion of two enzymatic indicators of renal damage, N-acetyl-beta-D-glucosaminidase (NAG) and alanine aminopeptidase (AAP) was measured in female Wistar rats at different ages. NAG and AAP are localized at different sites of the nephron and are released into the urine when kidney damage occurs. Total protein flow, urinary volume and creatinine flow were also determined. In a parallel experiment, the effect of aging on renal blood flow (RBF) and glomerular filtration rate (GFR) was examined in young (1.5-month) adult (3-month) and elderly (20-month) female rats. Clearance following a single injection of [131I]o-iodohippurate (hippuran, OIH) was used for the measurement of effective RBF and as an index of tubular cell function. [125I]Iothalamate (IOT) clearance was used to measure GFR. With advancing age, an increase in NAG and AAP urinary flow appeared. The increases in protein excretion were greater than and previous to those of enzyme excretion. It is shown that absolute RBF and GFR (ml/min) in old rats are greater than in young or adult animals. When absolute RBF or GFR was divided by kidney weight (ml/min/g) no clearance changes appeared in any age group studied; only when clearance was expressed in relation to body weight (ml/min/100 g), a decrease in RBF and GFR was evidenced. This indicates that the rate of increase of both RBF and GFR with age is similar to that of kidney weight and lower than that of body weight. The present findings indicate that urinary markers of renal injury increase with age, whereas GFR and RBF only decrease when expressed as clearance related to body weight.

N-acetyl-beta-D-glucosaminidase (NAG) and alanine aminopeptidase (AAP) excretion after acute administration of acetaminophen, salsalate and aspirin in rats.

The present study was performed to compare renal injury induced by high acute doses of both the nonacetylated salicylate, salsalate (SSA) and the acetylated salicylate, aspirin (ASA). As a marker of renal injury, urinary excretion of N-acetyl-beta-D-glucosaminidase (NAG) and alanine aminopeptidase (AAP) were measured after oral administration of 600 mg/kg of each drug. Other tests to detect renal injury were also performed. Serum salicylic levels after SSA or ASA were determined in a different experiment. Acetaminophen (APAP) was used as a standard of renal toxicity. Both drugs increase NAG and to a greater extent, AAP excretion. Proteinuria and polyuria appear after both drugs. Changes in urinary sodium and potassium were also shown. Our results support the view that both acetylated and non-acetylated salicylates induce renal injury at toxicological doses.