"Back to the Future": A New Look at Hydroxypropyl Beta-Cyclodextrins.

Since the discovery about 30 years ago (2-hydroxypropyl) beta-cyclodextrin, a highly soluble derivative of beta-cyclodextrin, has become an approved excipient of drug formulations included both in the United States and European Pharmacopoeias. It is recommended to use as solubilizer and stabilizer for oral and parenteral formulations. Recently, its pharmacological activity has been recognized in various diseases. The increasing applications require a closer look to the structure-activity relationship. As (2-hydroxypropyl) beta-cyclodextrin (HPBCD) is always a mixture of isomers with various degrees and pattern of hydroxypropylation, no wonder that the products of different manufacturers are often different. Several HPBCDs were compared applying a battery of analytical tools including thin layer chromatography, high performance liquid chromatography (HPLC), HPLC-mass spectrometry (MS), and matrix-assisted laser desorption MS. We studied how the average degree of substitution affects the aggregation behavior, the toxicity, and the solubilizing effect on poorly soluble drugs. We found that the products with low average degree of substitution are more prone to aggregation. The samples studied are nontoxic to Caco-2 cells and have low hemolytic activity. The solubility enhancement of poorly soluble drugs decreases or increases with increasing degree of substitution or shows a maximum curve depending on the properties of the guest.

Methyl-beta-cyclodextrins: the role of number and types of substituents in solubilizing power.

Methylated cyclodextrins (CDs) are effective solubilizers of poorly soluble organic compounds. In this work, we compared various methylated ß-CDs concerning their structure characterized by nuclear magnetic resonance spectroscopy, composition analyzed by HPLC and solubilizing capability by using model compounds such as cholesterol, fatty acids, furosemide, tamoxifen, and amiodarone. All the commercially available methylated ß-CDs are mixtures of various isomers and homologues except trimethyl ß-CD. The effects of the degree of methylation, the composition, as well as the influence of further derivatization with ionic groups were studied. The number of methyl groups in a CD ring should be around 14 to get the highest solubility for the included guest molecules. Although the distribution of isomers and related compounds has hardly any effect at constant degree of substitution, the introduction of amino and succinyl moieties on the CD ring adds ionic interactions to the hydrophobic interactions of the inclusion complex formation, which might result in synergic effect in solubilization.

Supramolecular proteoglycan aggregate mimics: cyclodextrin-assisted biodegradable polymer assemblies for electrostatic-driven drug delivery.

Self-assembled, noncovalent polymeric biodegradable materials mimicking proteoglycan aggregates were synthesized from inclusion complexes of cationic surfactants with γ-cyclodextrin and the natural anionic polymer hyaluronan. The amorphous structure of this ternary system was proven by X-ray diffraction and thermal analysis. Light-scattering measurements showed that there was a competition between hyaluronic acid and the surfactant for the cyclodextrin cavity. These self-assembled supramolecular matrices were loaded with both hydrophilic and lipophilic drug substances for dissolution studies. The release of the entrapped drugs was found to be controlled by cations in the surrounding media and by biodegradation. Slow drug release in an ion-free medium became faster in physiological salt solution in which the macroscopic polymer matrix was disassembled. In contrast, the enzymatic degradation of hyaluronan was hindered in the polymeric matrix. The supramolecular systems consisting of γ-cyclodextrin as a macrocyclic host, a cationic surfactant guest, and hyaluronic acid as the anionic polymer electrostatically cross-linked by the inclusion complex of the first two was found to be a novel drug-delivery system for the controlled release of traditional drugs such as curcumin and ketotifen and proteins such as bovine serum albumin.

Cannabinoid-mediated regulation of the hypothalamo-pituitary-adrenal axis in rats: age dependent role of vasopressin.

OBJECTIVE: Adaptation to stress is a fundamental component of life and the hypothalamo-pituitary-adrenocortical axis (HPA) plays a crucial role in it. The place of cannabinoid influence seems to be in the brain, especially where corticotropin releasing hormone and vasopressin (AVP) secreting neurons are located. The role of AVP is considered to be more important in young than in adult rats. Here we addressed the question if cannabinoid-mediated regulation of the HPA involves AVP and if there is any difference between young and adult rats in this process. METHODS: 10-day-old and adult AVP deficient Brattleboro rats were compared with their heterozygous littermates 1h after WIN 55,212-2 (6mg/kg i.p.) injection. RESULTS: In control animals the injection led to elevated adrenocorticotropin (ACTH) and corticosterone hormone levels at both ages without remarkable age difference in ACTH levels while all corticosterone levels of adults was approximately 10-times higher. The ACTH secretion of young AVP deficient rats failed to react to WIN 55,212-2 injection while their corticosterone levels were even higher than their littermates. In contrast in adult the role of AVP was diminished. CONCLUSIONS: We can conclude that the peripheral administration of cannabinoids leads to HPA axis stimulation, which process involves AVP at least in the young rats. The discrepancy between ACTH and corticosterone levels in young rats suggests an alternative adrenal gland regulatory pathway, which might be present in all studied animals. However, it comes to the front just in AVP deficient pups.

Gender-specific regulation of the hypothalamo-pituitary-adrenal axis and the role of vasopressin during the neonatal period.

Studies in arginine vasopressin (AVP)-deficient Brattleboro rats suggest that AVP is the predominant secretagogue during the perinatal period. Here we tested the hypothesis that congenital lack of vasopressin differentially modifies the stress reactivity of male and female rat pups. Vasopressin-producing (heterozygous, AVP+) and AVP-deficient (AVP-) Brattleboro rat pups of both genders were used. In 10-day-old pups, 24-h maternal separation and single, as well as repeated, ether inhalation induced remarkable adrenocorticotropin (ACTH) elevation only in AVP+ pups, supporting the role of vasopressin in hypothalamo-pituitary-adrenal (HPA) axis regulation. Surprisingly, the corticosterone elevations were even more pronounced in AVP- pups, suggesting the possibility of an ACTH-independent corticosterone-secretion regulation. In the case of maternal separation, both the plasma ACTH and corticosterone levels were higher in females than in males, while in case of ether inhalation only the ACTH levels were higher in females. Gender did not influence the stress reactivity or the effect of the genotype. We conclude that the gender of the pups did not profoundly influence HPA axis activity (the mechanism seems to be the same), but in contrast to the general view, we suggest that the females have a more active HPA axis than the males already during the neonatal period. However, the resting corticosterone elevation-well known in adult females- is missing.

Response of the adrenomedullary system to early postnatal stress in the Brattleboro rat.

The sympathetic-adrenomedullary system and the pituitary-adrenocortical axis are linked to each other by chemical signals transferring information between both endocrine systems. Here we addressed the question of whether the neuropeptide arginine vasopressin (AVP) is involved in this type of information transfer during early postnatal development. The impact of congenital absence of AVP on the endocrine stress response was investigated using the AVP-deficient Brattleboro rat. Under resting conditions, we failed measure a significant difference in plasma norepinephrine levels between 10-day-old AVP-deficient homozygous juveniles versus heterozygous AVP-producing littermates. Interestingly, repeated ether exposure resulted in a reduction of plasma epinephrine levels in both genotypes. In the adrenal, we detected increased levels of the epinephrine-synthesizing enzyme phenylethanolamine-N-methyltransferase (PNMT) after ether inhalation in vasopressin-deficient pups only. These data provide insight into the development of the regulation of stress-related epinephrine secretion during ontogenesis. Furthermore, our results imply that the congenital absence of AVP affects the synthesis of PNMT in response to defined stressor exposure.

WIN-55,212-2 chronically implanted into the CA3 region of the dorsal hippocampus impairs learning: a novel method for studying chronic, brain-area-specific effects of cannabinoids.

We report here that local hippocampal WIN-55,212-2 implants release this cannabinoid agonist for extended periods, the release is restricted to the implanted brain region and is behaviorally active. Radiolabeled WIN-55,212-2 was implanted bilaterally into the CA3 region of the dorsal hippocampus by means of fused silica capillaries. Significant amounts of the compound were released from the implants for at least 10 days. No labeled WIN-55,212-2 was detected in other brain regions, for example, the cortex, amygdala, thalamus, hypothalamus, and pons. In a separate experiment, radiolabeled WIN-55,212-2 was implanted chronically into the same hippocampal region, and rats were assessed 8 days later in the object-recognition test. In contrast to controls, rats implanted with WIN-55,212-2 were unable to differentiate familiar and unfamiliar objects. Object recognition was reinstated by the cannabinoid antagonist SR141716A, as rats implanted with both WIN-55,212-2 and SR141716A did not differ from controls. Thus, chronic hippocampal WIN-55,212-2 implants impaired recognition memory via the CB1 receptor. The memory-impairing effects of acute cannabinoid treatments are well known, but the effects of chronic treatments are controversial. The rate and magnitude of tolerance, however, have been shown to be brain-area specific and cell-type specific. Here we show that chronic hippocampal treatments impair memory, suggesting that no tolerance develops in the hippocampus towards the memory-impairing effects of cannabinoids. The data also suggest that chronic, brain-area-specific effects of cannabinoids can be studied by the novel method described here.

High-performance liquid chromatographic determination of 2-hydroxypropyl-gamma-cyclodextrin in different biological fluids based on cyclodextrin enhanced fluorescence.

A high-performance size exclusion chromatographic method with analyte enhanced fluorescence detection is described for the analysis of 2-hydroxypropyl-gamma-cyclodextrin (HPGCD) in different biological fluids. The principle of detection was the in situ complexation of 8-anilinonaphthalene-1-sulfonic acid (ANS) by HPGCD. When HPGCD eluted from the column the increased fluorescence was measured at excitation and emission wavelengths of 270 and 512 nm, respectively. Solid-phase extraction cleanup and concentration of samples resulted in higher than 78% recovery of HPGCD for each of the studied biological fluids. Some important details of the method development as well as the validation of the method for rabbit plasma, rabbit aqueous humour, monkey plasma and monkey urine are given. The limits of quantification varied between 1 and 10 nmol/ml (correspond to 1.5-15 microg/ml) depending on the biological matrix used. The method was successfully adapted in another laboratory proving that HPGCD had not absorbed into aqueous humour and plasma after topical application of HPGCD containing eye drop in rabbits.