Self-assembly of cyclodextrin complexes: detection, obstacles and benefits.

Cyclodextrins (CDs) are cyclic oligosaccharides that form water-soluble inclusion complexes of lipophilic molecules. They are commonly used as pharmaceutical excipients. Recently it has been observed that CDs and CD complexes self-assemble in aqueous solutions to form transient clusters, nanoparticles and small microparticles. The critical aggregation concentration (cac) of the natural αCD, ßCD and γCD in pure aqueous solutions is about 25, 8 and 9 mg/ml, respectively. The cac of 2-hydroxypropyl-ß-cyclodextrin (HPßCD), that consists of a mixture of isomers, in pure aqueous solutions is significantly higher or about 118 mg/ml. Formation of guest/ CD complexes can increase or decrease the cac value. Due to the transient nature of the CD clusters and nanoparticles they can be difficult to detect and their presence is frequently ignored. However, formation of such particulate matter in aqueous CD solutions can lead to erroneous analytical results and product rejections during drug manufacturing. On the other hand, they have also given rise to formation of novel drug delivery systems with exceptional properties.

Cyclodextrin complexes of a globular protein and a lipophilic oligopeptide: the effect of structure and physicochemical properties.

Cyclosporine A (CyA) is a lipophilic oligopeptide that has a very limited solubility in water of only 0.008 mg/ml at ambient temperature. It has the ability to form inclusion complexes with cyclodextrin (CD) whose complexes can self-assemble to form aggregates. We have previously developed eye drops with CyA/CD aggregates. Our aim was to study cyclodextrin complexes of lysozyme, a small polar globular protein, and to compare the results with those obtained for CyA. We also wanted to test the stabilizing effect of CDs on lysozyme. Phase-solubility studies of various CDs were performed with CyA and lysozyme. Complexation and particle size measurements were made with dynamic light scattering (DLS) and UV. Solid drug fractions were determined. Thermal and chemical stability studies were performed on lysozyme in the presence of various CDs. Recovery of lysozyme activity in the presence of various CDs after a heat shock was determined. Both CyA and lysozyme are able to form non-inclusion complexes with CD and those complexes can self-assemble and form micro sized aggregates. In case of lysozyme the forces involved are relativity weak and the lysozyme/CD complexes dissociate upon centrifuging, however for CyA the aggregates are stronger and do not dissociate upon centrifuging. CyA is therefore suitable for eye drop preparations containing CDs for sustained drug release whereas lysozyme is not. This is mainly due to the fact that CyA forms inclusion complexes with CDs, whereas lysozyme is not able to do so due to its polar surface. The lysozyme/CD non-inclusion complexes can offer some protection against lysozymes chemical and thermal denaturation. CD can, however, form complexes with unfolded lysozyme and hamper refolding of the protein after heat shock.

Effect of cyclodextrins on the degradation rate of benzylpenicillin.

The effect of cyclodextrin (CD) inclusion complexes on the degradation of benzylpenicillin in aqueous solutions was investigated at several different pH values and 37°C. The effects of neutral as well as both positively and negatively charged CDs were evaluated; all together 13 different CDs. Kinetic studies with HPßCD and RMßCD at pH ranging from 1.2 to 9.6 showed that CDs have stabilizing effect on the ß-lactam ring in aqueous acidic media but generally accelerated the hydrolytic cleavage of the ß-lactam ring in neutral and basic media. At physiologic pH (pH 7.4) quaternary ammonium CD derivatives (i.e., positively charged CD derivatives) have the highest catalytic effect, resulting in 6- to 18-fold enhancement of hydrolysis rate, while both the neutral methylated CDs had much less effect, resulting in 2- to 3-fold enhancement, and the negatively charged CD derivatives, resulting in only about 1.1- to 1.2-fold enhancement in the hydrolytic cleavage of the ß-lactam ring. Addition of water-soluble polymers to the aqueous reaction media containing CDs was shown to decrease the catalyzing effects of CDs on the ß-lactam hydrolysis.

Drug permeation through biomembranes: cyclodextrins and the unstirred water layer.

Surface of the living epithelia is covered with mucus, a gel-like fluid containing mainly water (90-98%) and mucin (2-5%). This aqueous gel-like matrix forms an unstirred water layer (UWL) creating an aqueous diffusion barrier that hampers drug permeation through mucosal barriers. Frequently, the UWL is the main drug permeation barrier, especially in the case of small-molecular-weight lipophilic drugs. Under such conditions drug permeation can often be enhanced by forming water-soluble drug/cyclodextrin complexes. Cyclodextrins enhance drug delivery through biomembranes by increasing drug permeation through the UWL, that is, by increasing the availability of dissolved drug molecules juxtaposed to the membrane surface. Cyclodextrins only enhance drug permeation when the UWL contributes to the permeation barrier. Cyclodextrins do not enhance drug permeation when the permeation resistance of the UWL is much less than that of the membrane itself. The effect depends also on the physicochemical properties of the drug. As a rule the best results are obtained for lipophilic drugs that are poorly soluble in water, that form water-soluble complexes with cyclodextrins and that possess, as dissolved drug molecules, relatively high permeability through lipophilic biomembranes. It is extremely important to optimize drug vehicles with regard to the amount of cyclodextrin. Too much or too little cyclodextrin will lead to less than optimal drug availability. Good understanding of cyclodextrin complexation and how cyclodextrin affect drug permeation through biomembranes is essential for successful development of cyclodextrin-containing drug formulations.

Aqueous solubility and true solutions.

To the naked eye there is no sharp boundary between true solutions, where solute molecules are fully dispersed in the solvent, and colloidal solutions, where the solute molecules form very small (diameter < 50 nm) water-soluble aggregates, and smaller aggregates (diameter < 5 nm) are not easily detected by light scattering. In some cases small aggregates can have higher affinity than the individual test molecules for some specific receptors, but most activity studies are based on interactions of individual test molecules with some specific receptor and, thus, it is more likely that aggregate formation will result in decreased apparent activity during high throughput screening (HTS) and false negative results. Furthermore, aggregate formation will influence the physicochemical properties of drugs, such as their aqueous solubility, chemical stability and partitioning. Formation of drug/cyclodextrin inclusion complexes can be used to mimic nonspecific drug-receptor interactions. Studies in aqueous cyclodextrin solutions have shown that practically insoluble drugs (solubility < 0.05 mg/ml) form small molecular aggregates and formation of such aggregates increases with decreasing drug solubility. Novel methods for solubility determinations, which can distinguish between individual solute molecules and small molecular aggregates, can possible improve the efficacy of HTS for new drug candidates.

Solubility and permeability of steroids in water in the presence of potassium halides.

Water forms a network of hydrogen bonded water molecules that gives liquid water unique physicochemical properties. Ions that affect the network structure, e.g. potassium halides, are known to either increase or decrease aqueous solubilities of drugs. Most biological membranes consist of hydrophilic exterior and a lipophilic interior. Mathematically they can be treated as two-layer membranes, i.e. a hydrophilic water layer that is referred to as unstirred water layer (UWL) and a lipophilic membrane. The purpose of this study was to investigate if and then how ions affect drug permeation through the UWL. The effects of potassium halides on the solubility and permeability of dexamethasone and hydrocortisone was investigated. The potassium halides had either increasing or decreasing effect on their aqueous solubility but did not have any effect on their permeability through UWL.

Topical drug delivery to the posterior segment of the eye: anatomical and physiological considerations.

Drug delivery to the posterior segment of the eye is important for potentially treating various disorders in retina, choroid, vitreous humor and optic nerve. Due to anatomic membrane barriers and the lacrimal drainage it can be quite challenging to obtain therapeutic drug concentrations in the posterior parts of the eye after topical drug administration. Since the membrane barriers cannot be altered with non-invasive methods invasive methods such as direct drug injection into the vitreous humor and subconjunctival, subtenons capsule or suprascleral injections are gaining popularity. However, invasive methods can cause discomfort for the patient and can also lead to complications that are even more serious than the disease being treated. Alternatively, novel ophthalmic formulations can be developed that specifically target topical drug delivery to the posterior segment of the eye. Anatomical and physiological barriers in the eye are reviewed as well as the theoretical model of passive drug diffusion from the eye surface into the eye. It is shown that enhanced drug delivery through conjunctiva/sclera to retina can be obtained by formulating lipophilic drugs as hydrophilic drug/cyclodextrin complex solutions. Optimization of the delivery system by formulating the drug as a low-viscosity aqueous drug/cyclodextrin complex suspension results in sustained high concentrations of dissolved drug in the tear fluid which further increases the targeted drug delivery to the posterior segment.

Studies on curcumin and curcuminoids XXXI. Symmetric and asymmetric curcuminoids: stability, activity and complexation with cyclodextrin.

A series of curcuminoids, including curcumin, were studied with the main focus on their solubility, phase-distribution, hydrolytic stability and photochemical stability in cyclodextrin (CD) solutions. Their radical scavenging properties were also briefly studied. All the investigated derivatives were more stable towards hydrolytic degradation in CD solutions than curcumin, and the general order of the stabilising effect was HPbetaCD>MbetaCD>>HPgammaCD. In contrast, the photochemical studies showed that curcumin is generally more stable than its derivatives. Solubility and phase-distribution studies showed that curcuminoids with side groups on the phenyl moiety have higher affinity for the HPgammaCD than for the betaCDs and that the relative affinity of the larger HPgammaCD cavity increases with the curcuminoid molecule size. The radical scavenging studies showed that curcumin is more active than the derivatives investigated and that the free phenolic hydroxyl group may be essential for the scavenging properties. This study also indicates that the two halves of the symmetric curcumin molecule act as two separate units and scavenge one radical each.

Investigation of soft long chain quaternary ammonium compounds as co-factors to enhance in vitro gene delivery.

The effect of soft long chain quaternary ammonium antibacterial agents on the in vitro gene delivery of a luciferase plasmid to COS-1 cell lines was investigated. Low concentrations of these compounds could be used to enhance gene delivery with Lipofectamine Plus.

Influence of aqueous diffusion layer on passive drug diffusion from aqueous cyclodextrin solutions through biological membranes.

Most drugs permeate biological membranes via passive diffusion and it is generally assumed that the main barrier is the lipophilic structure of the membrane. However, we have observed that the unstirred water layer adjacent to the membrane surface can in some cases be a barrier just as effective as the lipophilic membrane itself. Hydrophilic cyclodextrins can enhance drug delivery through biological membranes by increasing the availability of dissolved drug molecules immediate to the membrane surface, i.e. by increasing drug delivery through the unstirred water layer. Cyclodextrins and drug/ cyclodextrin complexes are, in most cases, unable to permeate lipophilic membranes. Thus, excess cyclodextrin, more than is needed to solubilize the drug in the aqueous exterior, will hamper drug delivery through biological membranes.

Marine lipids as building blocks for soft quaternary ammonium compounds and their antibacterial activity.

Environmental friendly antibacterial agents have to degrade relatively rapid to non-toxic and inactive products after they have had their desired effect. Environmental friendly quaternary ammonium agents were designed according to Bodor's soft drug approach and evaluated in vitro. Structure-activity relationship (SAR) studies showed that the antibacterial activity of a given soft agent will only be acceptable if its chemical stability is adequate to allow the agent to express its activity for sufficient duration of time. However, the studies also showed that increasing the lipophilicity of a chemically labile antibacterial agent could increase its potency. Two of the lipophilic quaternary ammonium antibacterial agents evaluated had minimum inhibitory concentration (MIC) against Staphylococcus aureus as low as 2 microg/ml and estimated degradation half-life less than 4 to 6 days at room temperature. Decreased MIC could only be obtained by increasing the degradation half-life of the agents.

Preparation of solid drug/cyclodextrin complexes of acidic and basic drugs.

One of the main obstacles in pharmaceutical applications of cyclodextrins is their increase of the formulation bulk. Even at maximum incorporation 500 mg of a solid drug/cyclodextrin complex will only contain between 50 and 125 mg of the drug, assuming a low molecular weight drug (MW 200 to 400 Dalton) and an average molecular weight cyclodextrin (MW about 1500 Dalton). In general, the complexation efficiency is low and consequently the complex powder contains a significant amount of empty cyclodextrin molecules. In the present study the complexation efficiency is increased by ionization of the drug molecule through addition of volatile acid (i.e. acetic acid) or base (i.e. ammonia) to the aqueous complexation media of basic or acidic drugs, respectively. The volatile acid or base was then removed during lyophilization and heating in a vacuum oven resulting in formation of solid cyclodextrin complexes of the unionized drug. Thus, the complexation efficiency was temporary increased by the ionization but then again decreased leading to formation of the thermodynamically unstable solid drug/cyclodextrin complexes. When dissolved the energy of the system was lowered by expelling the drug molecules from the cyclodextrin cavities resulting in formation of supersaturated drug solutions and ultimately precipitation of the drug.

Soft antibacterial agents.

Hard drugs have been defined as drugs that are biologically active and non-metabolizable in vivo. Soft drugs are defined as drugs, which are characterized by predictable and controllable in vivo destruction (i.e. metabolism) to form non-toxic products after they have achieved their therapeutic role. Quaternary ammonium compounds, such as benzalkonium chloride, are hard antibacterial agents. Their toxicity limits their usage in humans and animals, and their chemical stability limits their usage for general environmental sanitation. Furthermore, due to their stability they are prone to induce selective antimicrobial pressure and bacterial resistance. Soft analogs of the currently available hard antibacterial agents are less toxic. However, although the soft analogs have been shown to possess antibacterial activity in in vitro studies, it is likely that their in vivo activity will be hampered by their chemical instability.

The use of chemically modified cyclodextrins in the development of formulations for chemical delivery systems.

Retrometabolic drug design provides a highly useful and directed approach for identifying new drug candidates with improved therapeutic indices based on predictable/controlled metabolism and/or site-targeted delivery. In the process, formulation becomes an important and integral concern especially for brain-targeting chemical delivery systems (CDS) based on the need for appropriate dosage form stability, solubility and dissolution characteristics. Adjuncts that have been useful in this regard are chemically modified, water soluble cyclodextrin derivatives such a 2-hydroxypropyl-beta-cyclodextrin (HP beta CD). These starch-derived excipients can interact with drugs via dynamic complex formation resulting in a number of beneficial pharmaceutical effects including increased apparent water solubility and stability as well as improved aesthetic and excipient compatibility properties. This cyclodextrin is approved in a number of product in the US and world-wide. HP beta CD has contributed to the development and preclinical/clinical testing of a number of CDS including E2 (estradiol)-CDS, AZT (zidovudine)-CDS, DEX (dexamethasone)-CDS and a neuropeptide CDS based on an enkephalin derivative. In these contexts, HP beta CD provided for stable and water-soluble dosage forms intended for parenteral administration.

Cyclodextrins in topical drug formulations: theory and practice.

Cyclodextrins are cyclic oligosaccharides with a hydrophilic outer surface and a somewhat lipophilic central cavity. Cyclodextrins are able to form water-soluble inclusion complexes with many lipophilic water-insoluble drugs. In aqueous solutions drug molecules located in the central cavity are in a dynamic equilibrium with free drug molecules. Furthermore, lipophilic molecules in the aqueous complexation media will compete with each other for a space in the cavity. Due to their size and hydrophilicity only insignificant amounts of cyclodextrins and drug/cyclodextrin complexes are able to penetrate into lipophilic biological barriers, such as intact skin. In general, cyclodextrins enhance topical drug delivery by increasing the drug availability at the barrier surface. At the surface the drug molecules partition from the cyclodextrin cavity into the lipophilic barrier. Thus, drug delivery from aqueous cyclodextrin solutions is both diffusion controlled and membrane controlled. It appears that cyclodextrins can only enhance topical drug delivery in the presence of water.

Cyclodextrin solubilization of benzodiazepines: formulation of midazolam nasal spray.

The cyclodextrin solubilization of three benzodiazepines, i.e. alprazolam, midazolam and triazolam, was investigated. The cyclodextrin solubilization was enhanced through ring-opening of the benzodiazepine rings and ionization of the ring-open forms. Additional enhancement was obtained through interaction of a water-soluble polymer with the cyclodextrin complexes. The ring-opening was pH-dependent and completely reversible, the ring-open forms dominating at low pH but the ring-closed forms at physiologic pH. The ring-closed forms were rapidly regenerated upon elevation of pH. In freshly collected human serum in vitro at 37 degrees C, the half-life for the first-order rate constant for the ring-closing reaction was estimated to be less than 2 min for both alprazolam and midazolam. Midazolam (17 mg/ml) was solubilized in aqueous pH 4.3 nasal formulation containing 14% (w/v) sulfobutylether beta-cyclodextrin, 0.1% (w/v) hydroxypropyl methylcellulose, preservatives and buffer salts. Six healthy volunteers received 0.06 mg/kg midazolam intranasally and 2 mg intravenously, and blood samples were collected up to 360 min after the administration. Midazolam was absorbed rapidly reaching maximum serum concentrations of 54.3+/-5.0 ng/ml at 15+/-2 min. The elimination half-life of midazolam was 2.2+/-0.3 h and the absolute availability was 73+/-7%. All mean values+/-SEM.

Intranasal administration of midazolam in a cyclodextrin based formulation: bioavailability and clinical evaluation in humans.

Intranasal administration of midazolam has been of particular interest because of the rapid and reliable onset of action, predictable effects, and avoidance of injections. The available intravenous formulation (Dormicum i.v. solution from Hoffmann-La Roche) is however less than optimal for intranasal administration due to low midazolam concentration and acidity of the formulation (pH 3.0-3.3). In this study midazolam was formulated in aqueous sulfobutylether-beta-cyclodextrin buffer solution. The nasal spray was tested in 12 healthy volunteers and compared to intravenous midazolam in an open crossover trial. Clinical sedation effects, irritation, and serum drug levels were monitored. The absolute bioavailability of midazolam in the nasal formulation was determined to be 64 +/- 19% (mean +/- standard deviation). The peak serum concentration from nasal application, 42 +/- 11 ng ml-1, was reached within 10-15 min following administration and clinical sedative effects were observed within 5 to 10 min and lasted for about 40 min. Intravenous administration gave clinical sedative effects within 3 to 4 min, which lasted for about 35 minutes. Mild to moderate, transient irritation of nasal and pharyngeal mucosa was reported. The nasal formulation approaches the intravenous form in speed of absorption, serum concentration and clinical sedation effect. No serious side effects were observed.

Methazolamide 1% in cyclodextrin solution lowers IOP in human ocular hypertension.

PURPOSE: To formulate aqueous eye drops containing methazolamide 1% in cyclodextrin solution and to evaluate their effect on intraocular pressure (IOP) in a double-blind randomized trial in humans. Methazolamide, a carbonic anhydrase inhibitor (CAI), has been used in oral doses in the treatment of glaucoma but hitherto has not been successfully formulated in eye drops. In this study the effects of methazolamide are compared with those of dorzolamide (Trusopt). METHODS: Methazolamide 1% was formulated in a 2-hydroxypropyl-beta-cyclodextrin with hydroxypropyl methylcellulose in aqueous solution. Eight persons with ocular hypertension were treated with the methazolamide-cyclodextrin eye drops and eight persons with dorzolamide (Trusopt), both groups at dosages of three times a day for 1 week. IOP was measured before treatment was begun and on days 1, 3, and 8 at 9 AM (peak) and 3 PM (trough). RESULTS: After 1 week of treatment, the peak IOP in the methazolamide group had decreased from 24.4 +/- 2.1 mm Hg (mean +/- SD) to 21.0 +/- 2.0 mm Hg, which is a 14% pressure decrease (P: = 0.006). In the dorzolamide group, the peak IOP decreased from 23.3 +/- 2.1 mm Hg to 17.2 +/- 3.1 mm Hg, which is a 26% pressure decrease (P: < 0.001). On average, the IOP declined 3.4 +/- 1.8 mm Hg after methazolamide administration and 6.1 +/- 3.6 mm Hg after dorzolamide. CONCLUSIONS: Through cyclodextrin complexation, it is possible to produce topically active methazolamide eye drops that lower IOP. This is the first double-blind clinical trial that demonstrates the efficacy of the classic CAIs in eye drop formulation.