Molecular docking and inhibition studies of α-amylase activity by labdane diterpenes from Alpinia nigra seeds

Wide varieties of synthetic drugs are available for combating type 2 diabetes but are not free of associated side effects. Commercially available antidiabetic drugs are known to be potent inhibitors of α-amylase which reduce postprandial hyperglycaemia. Here, we have investigated Alpinia nigra seed extracts and the isolated two labdane diterpenes (I and II) for the α-amylase inhibitory activity. These labdane type diterpenes showed more promising inhibitory effects (IC₅₀ value 24.3 ± 2.05 and 15.167 ± 0.52 μM for compound I and II, respectively) against α-amylase than the standard inhibitor, acarbose. For both compounds, the mode of enzymatic inhibition was found to be non-competitive with K _i 13.303 ± 0.065 and 12.19 ± 0.099 μM, respectively. Molecular docking studies revealed that both I and II bind the human pancreatic α-amylase in the active site cleft similar to the acarbose. Among all the compounds under investigation, acarbose and compound II were found to have the highest MolDock and re-rank score. Further molecular dynamic simulation studies also supports the docking results obtained for both I and II. This is the first report on α-amylase inhibitory effect of the two labdane diterpenes with their potential candidature as future antidiabetic drugs of herbal origin.     

Daboia russelli venom hyaluronidase: purification, characterization and inhibition by β-3-(3-hydroxy-4-oxopyridyl) α-amino-propionic Acid

The present study describes the purification and characterization of a hyaluronidase (DRHyal-II) from Daboia/Vipera russelli venom and its inhibition by β-3-(3-hydroxy-4-oxopyridyl) α-amino-propionic acid, the mimosine. Gel permeation and ion exchange chromatography were employed to isolate DRHyal-II. The molecular mass by MALDITOF mass spectrometry was found to be 28.3 kDa. Single band in reduced SDS-PAGE suggested the monomeric nature. It was optimally active at pH 5.5 and at 37C and require 150 mM NaCl in the reaction mixture. It was specific to hyaluronan substrate and belongs to class-I or the neutral active enzymes. DRHyal-II was non-toxic by itself but, it potentiated the myotoxicity of VRV-PL-VIII myotoxin and hemorrhagic activity of hemorrhagic complex (HC). In in vitro experiments, mimosine inhibited the activity of DRHyal-II and the hyaluronidase activity of whole venom dose dependently. In in vivo experiments, mimosine inhibited the DRHyal-II potentiated myotoxicity of VRV-PL-VIII myotoxin and hemorrhagic activity of HC. The inhibition was due to the formation of DRHyal-II-mimosine inhibitory complex that resulted in significant structural changes at secondary and tertiary levels as evidenced by fluorescence emission and CD spectral studies. Hence, in this study an attempt was made to establish the possible role of hyaluronidase activity in the pathology of Daboia/Vipera russelli venom and the beneficial effects of its inhibition with special emphasis on the management of local toxicity.     

Partial characterization of β-glucosidase, β-xylosidase,and α-l-arabinofuranosidase from Jiangella alba DSM 45237 and their potential in lignocellulose-based biorefining

Jiangella alba DSM 45237 exhibited excellent extracellular β-glucosidase (1.03 ± 0.09 U/mL), β-xylosidase (16.29 ± 0.23 U/mL), and α-l-arabinofuranosidase (7.00 ± 0.09 U/mL) production in the growth media containing 15 g/L wheat straw pretreated with NaOH. The optimum temperature was 40 °C for β-glucosidase and β-xylosidase, whereas it was 50 °C for α-l-arabinofuranosidase. Among them, α-l-arabinofuranosidase was relatively stable at 60 and 70 °C. Enzymes showed maximum activity at pH 8.0. Enzymes, particularly β-glucosidase and α-l-arabinofuranosidase, were able to tolerate NaCl up to a final concentration of 12% (v/w). Among solvents, only ethanol and methanol increased the β-glucosidase activity. The majority of solvents did not significantly affect β-xylosidase activity but increased α-l-arabinofuranosidase activity. Except for phenol, other lignocellulose-derived compounds did not cause a significant activity loss in enzymes. Some of them, such as vanillic acid and acetic acid, have even increased the activity of enzymes. Hydrolysis of pretreated wheat straw using the crude enzyme from J. alba DSM 45237 released 160.9 mg/gds reducing sugars. Analysis of hydrolysis products with thin layer chromatography (TLC) showed that major products were 5C sugars. This is the first report related to the characterization of β-glucosidase, β-xylosidase, and α-l-arabinofuranosidase from J.alba DSM 45237 to date.     

Purification and characterization of Ts15, the first member of a new α-KTX subfamily from the venom of the Brazilian scorpion Tityus serrulatus

Voltage-gated potassium channel toxins (KTxs) are basic short chain peptides comprising 23-43 amino acid residues that can be cross-linked by 3 or 4 disulfide bridges. KTxs are classified into four large families: α-, β-, γ- and κ-KTx. These peptides display varying selectivity and affinity for K_v channel subtypes. In this work, a novel toxin from the Tityus serrulatus venom was isolated, characterized and submitted to a wide electrophysiological screening on 5 different subtypes of Na_V channels (Na_V1.4; Na_V1.5; Na_V1.6; Na_V1.8 and DmNa_V1) and 12 different subtypes of K_V channels (K_V1.1 - K_V1.6; K_V2.1; K_V3.1; K_V4.2; K_V4.3; Shaker IR and ERG). This novel peptide, named Ts15, has 36 amino acids, is cross-linked by 3 disulfide bridges, has a molecular mass of 3956 Da and pI around 9. Electrophysiological experiments using patch clamp and the two-electrode voltage clamp techniques show that Ts15 preferentially blocks K_V1.2 and K_V1.3 channels with an IC₅₀ value of 196 ± 25 and 508 ± 67 nM, respectively. No effect on Na_V channels was observed, at all tested concentrations. Since Ts15 shows low amino acid identity with other known KTxs, it was considered a bona fide novel type of scorpion toxin. Ts15 is the unique member of the new α-Ktx21 subfamily and therefore was classified as α-Ktx21.1.     

Presynaptic α2-adrenoceptors mediating inhibition of noradrenalh and 5-hydroxytryptamine release in rat cerebral cortex: further characterization as different α2-adrenoceptor subtynes

Summary In a previous investigation it was suggested that the ₂-adrenoceptors regulating ³H-noradrenaline (³H-NA) release and the ₂-heteroreceptors regulating the release of ³H-5-hydroxytryptamine (³H-5-HT) from rat cerebrocortex synaptosomes represent different subtypes of the ₂-adrenoceptor in that (–)-mianserin potently blocked the receptors sited on 5-HT terminals but was ineffective at the autoreceptors (Raiteri et al. 1983).In this work a number of ₂-adrenoceptor antagonists were tested against NA as an inhibitor of the K⁺ (15 mmol/l)-evoked release of ³H-NA or ³H-5-HT (in presence of 1 mol/l desipramine or citalopram, respectively) from superfused rat neocortex synaptosomes. The order of apparent affinity of the antagonists was: idazoxan ORG 20769 (2-amino-4-(1-methyl-1,2,3, 6-tetrahydropyridin-4-yl)-thiazole-5-carbonitrile (Z)-2-butenedioate (1:1) salt) ORG 20350 (5-chloro-4-(1-butyl-1,2,5,6-tetrahydropyridin-3-yl)-thiazole-2-amine (Z)-2-butenedioate (1:1) salt) ORG 20091 (5-chloro-4-(1-methyl -1,2,5,6-tetrahydropyridin -3- yl)-thiazole-2-amine (Z)-2-butenedioate (2:1) salt) at the ₂-autoreceptor and idazoxan ORG 20769 > ORG 20091 » ORG 20350 at the ₂-heteroreceptor. Prazosin (1 gmol/l) or AR-C 239 (1 gmol/l) (2-[2-[4-(o-methoxyphenyl)piperazine-l-yl]ethyl]-4,4-dimethyl-1,3(2H,4H)isoquinolinedione) were ineffective in both systems.Idazoxan and ORG 20769 had a comparable apparent affinity at the ₂-autoreceptor (pK_B values: 8.45 and 8.42, respectively) and at the heteroreceptor (pK_B values: 8.16 and 8.15, respectively). In contrast, ORG 20350 was about 14-fold less. potent than the two previous compounds at the autoreceptor (pKB = 7.30) whereas it was ineffective at the heteroreceptor when tested up to 3 mol/l (pK_B < 5.5). Experiments with electrically-stimulated (2 Hz; 2 ms; 24 mA) rat cerebral cortex slices confirmed the data with synaptosomes. ORG 20350 shifted to the right in a parallel manner the concentration-response curve of clonidine at the ₂-autoreceptors (pA₂ = 7.25). The sulphydryl alkylating agent N-ethyl-maleimide (NEM; 3 mol/l) which has been proposed to inactivate pertussis toxin sensitive G proteins, abolished the inhibition of both ³H-NA and ³H-5-HT release caused by the ₂-adrenoceptor agonist clonidine (0.3 mol/l) in hippocampus synaptosomes. The effect of exposure to NEM was not modified during protection experiments with idazoxan.The results lend further support to the proposed existence of functional ₂-adrenoceptor subtypes. It should be noted that the two pharmacologically distinct receptors here characterized are present in the same brain area and within the same animal species. They are sited on the axon terminals of different neurons. Their function appears that of inhibiting NA or 5-HT release, respectively. Both ₂-auto- and heteroreceptors are likely to be coupled to G proteins. According to the current nomenclature, the receptors do not seem to belong to the _2B subtype. However only one of them might be classified as _2A. Send offprint requests to M. Raiteri at the above address     

Quantitative analysis of neosalacinol and neokotalanol,another two potent α-glucosidase inhibitors from Salacia species,by LC-MS with ion pair chromatography

A quantitative analytical method for the highly polar sulfonium pseudo-sugar constituents neosalacinol (3) and neokotalanol (4), another two potent α-glucosidase inhibitors isolated from Ayurvedic traditional medicine Salacia species, was developed by employing an ion pair reagent upon chromatographic separation. The optimum conditions for separation and detection of these two constituents were achieved on an ODS column (3-μm particle size, 2.1-mm i.d. × 100 mm) with 5 mM undecafluorohexanoic acid–MeOH (99:1, v/v) as the mobile phase and using MS equipped with an electrospray ionization source. More than ten samples of Salacia from different origins were analyzed, and the results indicated that the assay was reproducible and precise and could be readily utilized for evaluation of α-glucosidase inhibitory activity of Salacia species. By combining this assay with the quantitative analytical method previously developed for salacinol (1) and kotalanol (2), a more precise and strict evaluation of α-glucosidase inhibitory activities of extracts from Salacia species (R = 0.959 for maltase and 0.795 for sucrase) was achieved.     

Purification, molecular cloning and functional characterization of HelaTx1 (Heterometrus laoticus): the first member of a new κ-KTX subfamily

Given their medical importance, most attention has been paid toward the venom composition of scorpions of the Buthidae family. Nevertheless, research has shown that the venom of scorpions of other families is also a remarkable source of unique peptidyl toxins. The κ-KTx family of voltage-gated potassium channel (VGPC) scorpion toxins is hereof an example. From the telson of the scorpion Heterometrus laoticus (Scorpionidae), a peptide, HelaTx1, with unique primary sequence was purified through HPLC and sequenced by Edman degradation. Based on the amino acid sequence, the peptide could be cloned and the cDNA sequence revealed. HelaTx1 was chemically synthesized and functionally characterized on VGPCs of the Shaker-related, Shab-related, Shaw-related and Shal-related subfamilies. Furthermore, the toxin was also tested on small- and intermediate conductance Ca(2+)-activated K(+) channels. From the channels studied, K(v)1.1 and K(v)1.6 were found to be the most sensitive (K(v)1.1 EC(50)=9.9±1.6 μM). The toxin did not alter the activation of the channels. Competition experiments with TEA showed that the toxin is a pore blocker. Mutational studies showed that the residues E353 and Y379 in the pore of K(v)1.1 act as major interaction points for binding of the toxin. Given the amino acid sequence, the predicted secondary structure and the biological activity on VGPCs, HelaTx1 should be included in the κ-KTX family. Based on a phylogenetic study, we rearranged this family of VGPC toxins into five subfamilies and suggest that HelaTx1 is the first member of the new κ-KTx5 subfamily.     

Structural basis of DNA topoisomerase II-α (Top2-α) inhibition: a computational analysis of interactions between Top2-α and its inhibitors

Topoisomerases are enzymes that resolve winding problem of DNA during cellular processes. Because of essential roles of these enzymes in maintenance of cell function, topoisomerases are important targets for cancer chemotherapy. To date, several topoisomerase inhibitors have been introduced and applied as drugs in the treatment of cancer. Topoisomerase II α (Top2-α), a subclass of topoisomerase II enzymes, functions as the target for several anticancer agents and a variety of mutations in this protein have been associated with the development of drug resistance. Mitoxantrone and Amsacrine are among two important inhibitors of Top2 enzymes used in cancer chemotherapy. In this study, we used computational methods to analysis interactions between these compounds and Top2-α in order to identify the most important residues involved in the enzyme inhibition. In order to obtain reliable results, several docking studies have been performed on the human Top2-β to reproduce binding modes which are observed in the crystal form of Top2-β complexed with Mitoxantrone and Amsacrine. Since human Top2-β is the closest homologue to Top2-α, same docking parameters have been used for docking of Top2-α with mentioned drugs. The data also showed that the main residues involved in the interaction between Top2-α and Mitoxantrone were Lys489, Asp504, Glu506, Gly488, Ile490 and Leu491. For Top2α-Amsacrine complex, the interaction was mainly through Arg487, Glu506, Gly488, Lys489, Asn504 and Ala505. These findings clarify the mechanisms of action for these drugs and may facilitate future drug development and cancer treatment.     

Hepatoprotective activity of berberine is mediated by inhibition of TNF-α, COX-2, and iNOS expression in CCl(4)-intoxicated mice

This study investigated the protective effects of isoquinoline alkaloid berberine on the CCl(4)-induced hepatotoxicity in mice. Berberine was administered as a single dose at 5 and 10mg/kg intraperitoneally (i.p.), 1h before CCl(4) (10%, v/v in olive oil, 2ml/kg) injection and mice were euthanized 24h later. The rise in serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP) in CCl(4)-intoxicated mice was markedly suppressed by berberine in a concentration-dependent manner. The decrease in hepatic activity of superoxide dismutase (Cu/Zn SOD) and an increase in lipid peroxidation were significantly prevented by berberine. Histopathological changes were reduced and the expression of tumor necrosis factor-α (TNF-α), cyclooxygenase-2 (COX-2), and inducible nitric oxide synthase (iNOS) was markedly attenuated by berberine 10mg/mg. The results of this study indicate that berberine could be effective in protecting the liver from acute CCl(4)-induced injury. The hepatoprotective mechanisms of berberine may be related to the free radical scavenging and attenuation of oxidative/nitrosative stress, as well as to the inhibition of inflammatory response in the liver.     

Effect of inhibition of γ-glutamyltranspeptidase by AT-125 (Acivicin) on glutathione and cysteine levels in rat brain and plasma

Summary AT-125 (Acivicin) is an inhibitor of -glutamyltranspeptidase (-GTP) which initiates glutathione catabolism to cysteine. We measured plasma and brain glutathione and cysteine in rats treated with AT-125. Six h after AT-125 treatment, plasma glutathione had increased 6-fold and plasma cysteine had fallen significantly. Brain cysteine fell after 24 h of AT-125 treatment, and brain glutathione had also decreased 18%. AT-125 pretreatment inhibited brain uptake of ³⁵S when it was given as ³⁵S-GSH but had no effect when it was given as ³⁵S-cysteine. These results suggest that plasma glutathione is catabolized by -GTP, and cysteine derived from it is taken up by the brain. N-acetylcysteine was administered to AT-125 treated rats in an attempt to supply cysteine to the brain in the face of -GTP inhibition. N-acetylcysteine supported brain glutathione levels, suggesting that it can serve as a source of cysteine under these conditions.Abbreviations -GTP -glutamyltranspeptidase - GSH reduced glutathione - TCA trichloroacetic acid     

Identification and in vivo and in vitro characterization of long acting and melanocortin 4 receptor (MC4-R) selective α-melanocyte-stimulating hormone (α-MSH) analogues

We report in vitro and in vivo data of new α-melanocyte-stimulating hormone (α-MSH) analogues which are N-terminal modified with a long chain fatty acid derivative. While keeping the pharmacophoric motif (d-Phe-Arg-Trp) fixed, we tried to improve selectivity and physicochemical parameters like solubility and stability of these analogues by replacing amino acids further away from the motif. Receptor specific changes in binding affinity to the melanocortin receptors were observed between the acetyl derivatives and the fatty acid analogues. Furthermore, amino acids at the N-terminal of α-MSH (Ser-Tyr-Ser) not considered to be part of the pharmacophore were found to have an influence on the MC4/MC1 receptor selectivity. While the acetyl analogues have an in vivo effect for around 7 h, the long chain fatty acid analogues have an effect up to 48 h in an acute feeding study in male Sprague-Dawley rats after a single subcutaneous administration.     

Disposition of (—)-fenfluramine and its active metabolite, (—)-norfenfluramine in rat: A single dose-proportionality study

1. The disposition of (—)-fenfluramine, (—)-F, was studied in rats after i.v. and oral administration (1˙25 to 12˙5 mg/kg). Whole blood-to-plasma ratio and the protein binding (determined by equilibrium dialysis) of the compound and its main active metabolite, (—)-norfenfluramine (—)-NF, were investigated.

2. The bound fraction of both compounds (about 40%) was constant in the concentration range of 1-10 nmol/ml. The whole blood to plasma concentration ratios of (—)-F and (—)-NF were larger than unity and were constant over this dose range.

3. The drug followed apparent first-order kinetics, at doses up to 6˙25 mg/kg. The mean half-lives of the parent drug and its metabolite were about 1 and 12 h respectively. The volume of distribution of (—)-F was large and total body clearance approached liver blood flow.

4. Oral doses were rapidly absorbed from the rat gastrointestinal tract. Bioavailability of the drug was about 20%. Urinary excretion of unchanged drug (3-4% of dose) and its metabolite (about 20%) were similar after i.v. and oral administration.

5. After larger doses (12˙5 mg/kg) the kinetics of (—)-F were nonlinear. The AUC increased, but not in proportion to the dose, and kinetic parameters were modified.

6. Brain concentrations reflected the dose-related changes observed in (—)-F and (—)-NF blood concentrations, and patterns of brain distribution and subcellular localization of the drug and its metabolite were modified at the highest dose tested.     

Donepezil (E2020): a new acetylcholinesterase inhibitor. Review of its pharmacology,pharmacokinetics, and utility in the treatment of Alzheimer’s disease

Donepezil is an acetylcholinesterase inhibitor under development for the treatment of mild-moderately Alzheimer's disease. In vitro, donepezil is about 10 times more potent than tacrine as an inhibitor of acetylcholinesterase. Donepezil is 500 - 1000 fold selective for acetylcholinesterase over butyrylcholinesterase. In animal models, donepezil produces positive effects on both working memory and long term memory. In man, donepezil is slowly absorbed from the gastrointestinal (GI) tract. The compound has a terminal elimination half-life of 50 - 70 h in young volunteers; in elderly volunteers, the half-life of the compound is extended to over 100 h. Donepezil is extensively metabolised after oral administration. The parent compound is 93% bound to plasma proteins. Results from two clinical trials with donepezil were published. The largest of these trials was a 12 week 161 patient Phase II investigation in the USA. Results from this investigation showed that donepezil produced dose-related improvements, with statistically significant effects occurring at doses of 3 and 5 mg/day. The results published to date suggest that donepezil will be a useful agent in the symptomatic treatment of Alzheimer’s disease.     

Facilitation of serotonin (5-HT) release in the rat brain cortex by cAMP and probable inhibition of adenylate cyclase in 5-HT nerve terminals by presynaptic α2-adrenoceptors

Summary Stimulation-evoked tritium overflow was examined in superfused rat brain cortex slices (stimulus: electrical impulses; 3 Hz) and synaptosomes (stimulus: potassium 12 mmol/l) preincubated with ³H-5-HT. 1. In slices and synaptosomes, the evoked ³H overflow was facilitated by forskolin and 8-Br-cAMP, but was not affected by AH 21-132 (an inhibitor of cAMP phosphodiesterase; cis-6-(p-acetamidophenyl)-1, 2, 3, 4, 4 a,10b-hexahydro-8, 9-dimethoxy-2-methylbenzo [c] [1,6]-naphthyridine). In the presence of AH 21-132, the facilitatory effect of forskolin on evoked overflow was increased. 2. In slices, AH 21-132 or combined administration of forskolin plus AH 21-132 did not change the percentage of basal or evoked ³H overflow represented by unmetabolized ³H-serotonin (about 30% and 60%, respectively). 3. In slices, cocaine or 6-nitroquipazine, an inhibitor of serotonin uptake, did not influence the increase in evoked overflow produced by forskolin plus AH 21-132. Forskolin plus AH 21-132 did not alter the inhibitory effect of serotonin (examined in the presence of 6-nitroquipazine) and the facilitatory effect of metitepin (a serotonin receptor antagonist) on evoked ³H overflow, but considerably decreased the inhibitory effect of clonidine or B-HT 920 (2-amino-6-allyl-5,6,7,8-tetrahydro-4H-thiazolo-[5,4-d]-azepine). The present results suggest that the serotoninergic nerve terminals in the rat brain cortex are endowed with an adenylate cyclase, which is negatively coupled to the presynaptic ₂-adrenoceptors, but is not linked to the presynaptic autoreceptors.This study was supported by a grant of the Deutsche Forschungsgemeinschaft. Part of the present results was reported at the 9th International Congress of Pharmacology, London 1984 (Schlicker et al. 1984) Send offprint requests to M. Göthert     

New 1,2,3-triazole–(thio)barbituric acid hybrids as urease inhibitors: Design,synthesis, in vitro urease inhibition,docking study,and molecular dynamic simulation

A new series of 1,2,3-triazole–(thio)barbituric acid hybrids 8a – n was designed and synthesized on the basis of potent pharmacophores with urease inhibitory activity. Therefore, these compounds were evaluated against Helicobacter pylori urease. The obtained result demonstrated that all the synthesized compounds, 8a – n , were more potent than the standard urease inhibitor, hydroxyurea. Moreover, among them, compounds 8a , 8c – e , 8g , h , and 8k , l exhibited higher urease inhibitory activities than the other standard inhibitor used: thiourea. Docking studies were performed with the synthesized compounds. Furthermore, molecular dynamic simulation of the most potent compounds, 8e and 8l , showed that these compounds interacted with the conserved residues Cys592 and His593, which belong to the active site flap and are essential for enzymatic activity. These interactions have two consequences: (a) blocking the movement of a flap at the entrance of the active site channel and (b) stabilizing the closed active site flap conformation, which significantly reduces the catalytic activity of urease. Calculation of the physicochemical and topological properties of the synthesized compounds 8a – n predicted that all these compounds can be orally active. The ADME prediction of compounds 8a – n was also performed.     

Interaction of [d-Trp6, Des-Gly10] LHRH Ethylamide and Hydroxy Propyl β-Cyclodextrin (HPβCD): Thermodynamics of Interaction and Protection from Degradation by α-Chymotrypsin

Purpose: The purpose of this study is to investigate the mechanisms and thermodynamics of the interaction between hydroxypropyl β-cyclodextrin (HPβCD) and [d-Trp ⁶, des-Gly ¹⁰] LHRH ethylamide (deslorelin), a peptide drug. Methods: We used UV and Fluorescence spectroscopy to study the interaction of HPβCD and deslorelin. Circular dichroism was used to study the conformational changes induced in deslorelin upon interaction with HPβCD. The thermodynamics of the interaction of deslorelin and HPβCD was studied using isothermal titration calorimetry (ITC). We also determined the effect of HPβCD on the degradation of deslorelin by α-chymotrypsin. Results: UV and fluorescence spectroscopy indicated that HPβCD induced a change in polarity of the environment surrounding the chromophores of deslorelin. Wavelength selective fluorescence indicated an increase in the fluorescence polarization of deslorelin with an increase in excitation wavelength in the presence of HPβCD suggesting that tryptophan is present in a media of reduced mobility. Circular dichroism studies indicated that HPβCD stabilizes the conformation of deslorelin. In addition, ITC indicated an exothermic reaction between deslorelin and HPβCD with a low enthalpy of binding of ～?600 cal/mol and a binding affinity of ～1.25 × 10 ² M^{? 1}. Finally, the rate of degradation of deslorelin by α-chymotrypsin was decreased by 33% in the presence of HPβCD. Conclusions: These results indicate that there is an interaction between HPβCD and deslorelin, which involves the inclusion of aromatic amino acids of deslorelin into the hydrophobic cavity of the cyclodextrin. This inclusion, providing steric hindrance, may be one of the mechanisms by which HPβCD reduces enzymatic hydrolysis of deslorelin.