Marcus E. Brewster

Use of 2-hydroxypropyl-beta-cyclodextrin as a solubilizing and stabilizing excipient for protein drugs.

A chemically modified, amorphous β-cyclodextrin, namely, 2-hydroxypropyl-β-cyclodextrin (HPCD), was examined as a solubilizing and stabilizing agent for protein drugs. The aqueous solubility of ovine growth hormone at pH 7.4 was increased through the use of HPCD. This effect was manifested by higher UV transparency at 600 nm. Interleukin-2 (IL-2) is rendered insoluble upon lyophilization in the absence of stabilizers. Use of aqueous HPCD provides a clear solution, as indicated by fluorometric light scattering, and inhibits aggregate formation, as shown by ultracentrifugation and Western blot analyses. In addition, there were no major conformational changes of IL-2 in HPCD formulation as indicated by fourth-derivative ultraviolet spectroscopy. Finally, IL-2 retained 100% of its biopotency when prepared in HPCD solutions. Aggregation of insulin was also suppressed by HPCD. These data, as well as the i.v. safety of HPCD and its well-characterized chemical composition, suggest that this starch derivative may be a potentially useful excipient for protein drugs intended for parenteral use.

An intravenous toxicity study of 2-hydroxypropyl-β-cyclodextrin, a useful drug solubilizer, in rats and monkeys

Abstract The subacute and subchronic intravenous (i.v.) toxicity of 2-hydroxypropyl-β-cyclodextrin (HPCD) was examined in Sprague-Dawley rats and cynomolgus monkeys. After either 14 or 90 days of alternate day dosing with either saline or 200 mg/kg HPCD, no toxicologically meaningful differences were observed in the following parameters: body weight, body weight gain, food consumption, hematology, clinical chemistry, organ weights, organ to brain weight ratio, organ to body weight ratio and macroscopic and microscopic histopathology. Subsequent to these studies, an acute high-dose study was performed in cynomolgus monkeys which indicated that a single i.v. dose of HPCD as high as 10 g/kg was not lethal.

Effect of various cyclodextrins on solution stability and dissolution rate of doxorubicin hydrochloride

Abstract A number of cyclodextrins were examined as to their ability to stabilize doxorubicin in solution and to enhance the rate of its dissolution. 2-Hydroxypropyl-β-cyclodextrin (HPβCD), 2-hydroxypropyl-γ-cyclodextrin (HPγCD) and γ-cyclodextrin (γCD) all acted to decrease degradation of doxorubicin in solution although the γ-cyclodextrin derivatives were decidedly more effective. Lineweaver-Burk analysis indicated that the incorporated anthracycline degraded 6–10-times more slowly than did doxorubicin in systems which did not contain Cyclodextrin. The chemically modified cyclodextrins were also shown to enhance the rate of dissolution of prototype lyophilized formulations. Thus, HPγCD, present in 5-fold excess relative to doxorubicin, acted to decrease dissolution time from 26 min (for a doxorubicin/lactose dosage form) to less than 9 min. Similarly, HPγCD decreased dissolution time for various prototype formulations. Finally, a complex of HPγCD and doxorubicin was found to be less toxic in a dermal extravasation model. These results, along with the accrued toxicity data which suggest HPγCD is innocuous even when given in large doses parenterally, highly support the use of the modified Cyclodextrin as a stabilizing and dissolution-enhancing excipient.

Improved delivery through biological membranes. XXII. Synthesis and distribution of brain-selective estrogen delivery systems

Abstract 1,4-Dihydrotrigonelline derivatives of the estrogens, estradiol (E 2 ), estrone (E 1 ) and estradiol-3-methyl ether (3 MeE 2 ) were synthesized as potential brain-selective delivery forms for these steroids. Initial in vivo screening showed that the estradiol chemical delivery system (E 2 CDS) was most appropriate for further in vivo study. Accordingly, when E 2 CDS was systemically administered to rats, sustained levels of the oxidized, trigonelline ester delivery system (E 2 Q + ) were found in the brain while peripheral levels of E 2 Q + rapidly fell. In addition, radioimmunoassay (RIA) and spectroscopic methods detected E 2 in the brain after administration of E 2 CDS. The undetectable levels of E 2 in the blood suggest that the E 2 found in the central nervous system (CNS) was derived from centrally delivered E 2 Q + and not from peripheral sources.

A NON-SURFACTANT FORMULATION FOR ALFAXALONE BASED ON AN AMORPHOUS CYCLODEXTRIN : ACTIVITY STUDIES IN RATS AND DOGS

Abstract The steroid anesthetic, alfaxalone, is commercially available for veterinary use in a formulation containing Cremophor-EL® and alfadalone acetate. The allergic sensitivity of some species to this formulation limits its veterinary application and also led to withdrawal of the product for human use. The present studies evaluated alfaxalone formulated as an inclusion complex with a mixture of 2-hydroxypropyl-β-cyclodextrin (HPCD) isomers for anesthetic properties in rats and dogs. Aqueous solutions of alfaxalone-HPCD showed identical anesthetic profiles in rats compared to the commercial formulation. In contrast, the anesthetic response to alfaxalone-HPCD in dogs was not accompanied with massive histamine release and pronounced decrease in respiration and blood pressure seen after the commercial preparation. Anesthesia induction and recovery phases were rapid and uncomplicated after alfaxalone-HPCD. An anaphylactoid-like response did occur in dogs after the same dose of alfaxalone as the commercial preparation. The favorable anesthetic profile of alfaxalone formulated as an HPCD complex together with previous toxicology and stability studies suggest that the steroid may be successfully formulated for intravenous dosing with modified cyclodextrins.

The use of 2-hydroxypropyl-β-cyclodextrin as a vehicle for intravenous administration of dexamethasone in dogs

Abstract Dexamethasone (Dex) was administered to 6 mongrel dogs in a dose of 5 mg/kg as the sodium salt of its phosphate ester or as an inclusion complex in 2-hydroxypropyl-β-cyclodextrin (HPCD). Blood and urine were collected and analyzed for dexamethasone by HPLC. The area under the plasma concentration time curve (AUC) of dexamethasone during the first hour was significantly greater after the administration of the Dex-HPCD complex compared to the phosphate ester prodrug (1.63 ± 0.13 vs 1.04 ± 0.10 μ g h ml −1 ). In addition, the renal clearance (CL ren ) was significantly higher after administration of the HPCD form (0.91 ± 0.20 vs 0.52 ± 0.20 ml min −1 kg −1 ). There was no difference between treatments for other pharmacokinetic parameters of dexamethasone. Hence, intravenous injection of dexamethasone in HPCD allows direct administration of this poorly soluble steroid without any prodrug conversion. The higher plasma levels during the first hour after administration may be advantageous for the treament of emergency situations.

A novel redox system for CNS-directed delivery of estradiol causes sustained LH suppression in castrate rats

A series of 4 studies was conducted to examine the estrogen-like activity of a chemical delivery system (CDS) coupled to estradiol (E2). The CDS is based on a redox system, analogous to the NAD+ in equilibrium NADH coenzyme system and has previously been shown capable of sustained and site specific drug delivery to the central nervous system. The ability of CDS-E2 to suppress luteinizing hormone (LH) in gonadectomized rats was examined as an index of sustained estrogen action. A single dose of CDS-E2 resulted in significantly decreased LH serum levels in castrate rats through at least 24 days while an equimolar dose of E2 resulted in only transient LH decrease. Serum E2 levels were not different between the treatment groups, indicating that peripheral estrogen could not readily explain sustained hormone activity. A dose-response relationship was observed 12 days post-drug treatment in all monitored estrogen activities which showed CDS-E2 is more potent compared to equimolar E2. Further, LH suppression was significantly greater compared to ovariectomized rats treated with equimolar estradiol valerate, while anterior pituitary weights were not different between groups. Together with our previous data, these studies show that CDS-E2 exerts sustained estrogen-like activity which cannot be readily attributed to circulating E2 levels. These findings are consistent with a sustained, brain directed delivery of estrogen.

High-Performance Liquid Chromatographic Assay of a Central Nervous System (CNS)-Directed Estradiol Chemical Delivery System and Its Application After Intravenous Administration to Rats

A redox-based chemical delivery system for estradiol (E2-CDS) has been shown capable of sustained and brain-selective delivery of estradiol (E2). A re versed-phase high-performance liquid chromatographic (HPLC) method is presented for the analysis of E2-CDS and its oxidized quaternary metabolite (E2-Quat) in biological fluids or tissues. The assay utilizes a precolumn enrichment technique and detects plasma levels down to 10 ng/ml E2-Quat and 20 ng/ml E2-CDS. Sample preparation is rapid and simple. Samples are homogenized with acetonitrile, then centrifuged, and the supernatant is directly injected into the HPLC system. A water delivering pump injects the sample on a precolumn where the drug is concentrated. The mobile phase backflushes the retained compound onto the analytical column. At the same time, another sample can be injected onto a second precolumn. This alternating precolumn sample enrichment technique allows the injection of large volumes, up to 1800 µl. Plasma and tissue samples of rats collected after i.v. administration of a single 15-mg/kg E2-CDS dose were analyzed for E2-CDS and E2-Quat by this procedure. The results show sustained brain levels of E2-Quat and prolonged half-life in brain compared to six peripheral tissues measured. These data support the concept of brain-targeted delivery using redox carrier systems of this type.

Chronic weight loss in lean and obese rats with a brain-enhanced chemical delivery system for estradiol

Studies were undertaken to determine the effects on body weight and food intake of a chemical delivery system which preferentially delivers estradiol (E2) to the brain and there serves as a source for the sustained release of the steroid. We injected intravenously various doses of this estradiol-chemical delivery system (E2-CDS), E2-valerate (E2-VAL) or the dimethyl sulfoxide (DMSO) vehicle to young lean male rats and monitored body weight and 24 hr food intake for 39 days postinjection. E2-VAL caused a transient reduction in food intake and body weight gain. By contrast, a single injection of E2-CDS caused a chronic, dose-dependent reduction in the rate of body weight gain. In these lean rats, the duration of reduced body weight gain was not correlated with the observed transient reduction in food intake. In aged, obese male rats, E2-CDS caused a marked and chronic dose-dependent reduction in body weight. In contrast to lean rats, E2-CDS caused a long-term reduction in food intake in obese rats. To evaluate the importance of the E2-CDS-induced reduction in food intake in the observed persistent weight loss in obese rats, E2-CDS was administered to a group of obese rats and a second group which received the DMSO vehicle was pair-fed an equivalent amount of food daily. The resulting weight loss in both groups was equivalent. These results show that the enhanced delivery of E2 to the brain with the E2-CDS causes sustained reduction in the rate of body weight gain in lean rats and persistent weight loss in obese animals.

A dihydropyridine conjugate which generates high and sustained levels of the corresponding pyridinium salt in the brain does not exhibit neurotoxicity in cynomolgus monkeys

Many drugs can be selectively delivered to the brain by using a dihydropyridine in equilibrium pyridinium salt chemical delivery system (CDS). The interaction of these systems with central dopaminergic function was examined in this communication. Castrate female Sprague-Dawley rats when treated with a CDS for estradiol (i.e. 3-hydroxy-17 beta-[( (1-methyl-1,4-dihydropyridin-3-yl)carbonyl]oxy) estra-1,3,5(10)-triene or E2CDS) exhibit sustained and profound suppression of serum levels of leuteinizing hormone (LH). Treatment of rats with pargyline (80 mg/kg) prior to E2CDS (2 mg/kg) did not mitigate the biological effectiveness of this estrogen indicating at least indirectly that monoamine oxidate (MAO) was not involved in the CDS activation. In a more direct examination, cynomolgus monkeys treated with various repeated doses of E2CDS (cumulative doses of 0.2-40.0 mg/kg) demonstrated neither impaired motor function nor depleted striatal dopamine concentrations. The latter parameter was measured using liquid chromatographic-electrochemical analysis. These experiments support the contention that the CDS is not neurotoxic and further strengthens the strict structure-activity requirements for MPTP-induced neurotoxicity.