Cyclodextrins in oligonucleotide delivery.

The aim of this contribution is to summarize recent findings on the potential use of cyclodextrins and their derivatives as carriers for oligonucleotide agents. Their peculiar properties could be exploited in such an emerging therapeutic area by virtue of their capability of interacting with cellular membranes, thus giving rise to improved cellular uptake. In particular, some specific derivatives could be considered as promising future excipients for the delivery of "naked" antisense and/or decoy oligonucleotides which are difficult to formulate with existing pharmaceutical excipients.

Safety of parenteral hydroxypropyl beta-cyclodextrin.

Post-treatment data were collected on a patient who received intravenous hydroxypropyl beta-cyclodextrin in a dose of 1.5 g/kg in 1985. Although no untoward effects were observed in this patient, rarely occurring agitation and pulmonary edema have been noted after injections into rabbits and dogs, respectively. These complications are analyzed here on the basis of symptoms and on the effects of hydroxypropyl beta-cyclodextrin on the biochemistry of a representative lipid, cholesterol, which were studied in rats. It is hypothesized that these untoward effects of parenteral hydroxypropyl beta-cyclodextrin are due to complex formation, with lipid mediators of pathological responses, of which prostaglandins are one example. These mediators normally have brief and localized functions; if hydroxypropyl beta-cyclodextrin happens to be injected when these mediator systems are activated, their influence and the responses of the organism may be increased.

Parenteral hydroxypropyl cyclodextrins: intravenous and intracerebral administration of lipophiles.

Hydroxypropyl cyclodextrins are nontoxic carbohydrate derivatives of moderate molecular weight (1030-1750 Da) which form water-soluble complexes with many lipophiles. The fate of hydroxypropyl beta-cyclodextrin alone and in complex with testosterone or cholesterol injected intravenously or intracerebrally into rats was followed. More than 90% of intravenously administered hydroxypropyl beta-cyclodextrin was cleared into urine in 4 h, as previously described (Monbaliu, J.; Van Beijsterveld, L.; Meuldermans, W.; Szathmary, S.; Haykants, J. Abstracts, 5th International Symposium on Cyclodextrins, Paris, 1990; Abstract 65). After the injection of steroids in complex with hydroxypropyl beta-cyclodextrin into the tail vein of rats, the steroid component was released from the complex, before it reached the kidneys; the release occurred mainly into the proteins and lipoproteins of serum. Hydroxypropyl beta-cyclodextrins injected alone into the brain were cleared within less than 24 h, presumably via the flow of interstitial and cerebrospinal fluids, and eventually were excreted in urine. Testosterone, incorporated in a hydroxypropyl beta-cyclodextrin complex, after intracerebral injection was cleared from the brain even more rapidly than hydroxypropyl beta-cyclodextrin, presumably by crossing the blood-brain barrier and later removal to the liver by the specific carrier proteins in serum. Complexed cholesterol, in a similar experiment, was largely retained in the brain and its distribution there was uneven and remained that way for at least 3 days. It is clear that lipophilic agents, after their incorporation into hydroxypropyl beta-cyclodextrin complexes and subsequent in vivo administration, are rapidly released and exchanged into the plasma. In absence of plasma they enter tissues surrounding the injection site and thus are also promptly transferred into the organism's lipid systems. The manner in which different lipophilic agents are transported in vivo appears not to be greatly affected by their previous complexation; rather hydroxypropyl cyclodextrins just enable their entry in a larger amount and in an exchangeable, nonaggregated form.

Safety of oral cyclodextrins: effects of hydroxypropyl cyclodextrins, cyclodextrin sulfates and cationic cyclodextrins on steroid balance in rats.

Derivatives of beta-cyclodextrin differing in the length of a hydroxyalkyl substituent (CH2CH2OH, CH2CHOHCH3, CH2CHOHCH2CH2CH2CH3) or in the electrical charge of the substituents (SO4-, CH2CHOHCH2N(CH3)3+) and hydroxypropyl derivatives (CH2CHOHCH3) of alpha-, beta-, and gamma-cyclodextrin were compared, individually and in mixtures, as solubilizers of cholesterol. The most effective solubilizer proved to be hydroxypropyl derivatives of beta-cyclodextrin; beta-cyclodextrin sulfate (SO4-) was practically devoid of solubilizing activity. Oral administration of these cyclodextrin derivatives, some of which are both nondegradable and effective complexation agents for cholesterol and bile acids, nevertheless did not affect the conversion of [14C]acetic acid to [14C]-cholesterol in rat under the same conditions when another bile acid complexation agent, cholestyramine, increased that conversion. Thus, complexation of cholesterol and of bile acids by cyclodextrin derivatives, which is a significant and well-defined phenomenon in vitro, seems to have limited importance in terms of excretion of cholesterol from the gastrointestinal tract. It is proposed that various untoward effects observed after chronic large oral doses of hydroxypropyl beta-cyclodextrin are administered are not caused by an increased excretion of some vital lipophile or enzyme but are probably caused by solubilization and increased absorption of toxic contaminants of the ingested food.

Suppositories containing cyclodextrin complexes. Part 2: Dissolution and absorption studies.

The dissolution and absorption of poorly water-soluble drugs from rectal suppositories can be enhanced by complexing these substances (e.g., essential oils, indomethacin) with beta-cyclodextrin. Preliminary in vivo studies showed, that the application of cyclodextrin complexes to suppositories, the same as to oral applications, resulted in an increased blood level.

Absorption, distribution, excretion and metabolism of orally administered 14C-beta-cyclodextrin in rat.

The absorption, distribution, excretion and metabolism of orally administered universally labelled 14C-beta-cyclodextrin and 14C-glucose were compared in rat. The maximum radioactivity of the blood derived from 14C-beta-cyclodextrin was observed between 4th and 11th h and the value of the maximum in different experiments ranged between 5 and 17 0/00 of the total administered radioactivity. Following 14C-glucose treatment radioactivity reached the maximum within half-an-hour, with values of 15 to 82 0/00. In the 8th h after a high dose (313.5 mg/kg) of beta-cyclodextrin no more than 3-50 ppm beta-cyclodextrin was detectable in the blood by HPLC. After 14C-beta-cyclodextrin treatment 4.2-4.8% of the administered total radioactivity was excreted by the urine and about the same quantity (2-3.6%) in case of 14C-glucose. No specific accumulation was observed after 14C-beta-cyclodextrin treatment in the different organs. The large intestine contained 10-15% of the cyclodextrin radioactivity while this value was only 2% in case of 14C-glucose. Following p.o. administration of different doses of 14C-beta-cyclodextrin the radioactivity peak was detected in the exhaled air between the 4-6th and 6-8th h, respectively, depending on the administered doses, while in case of 14C-glucose treatment it was observed within 2 h. The total radioactivity exhaled by 14C-beta-cyclodextrin treated animals in 24 h was 55 to 64% of the administered radioactivity and 58% in case of 14C-glucose. It is assumed that beta-cyclodextrin is metabolized in rats slower but similarly to glucose, therefore p.o. administered beta-cyclodextrin cannot induce toxic symptoms.

Improvement of the absorption of 3H-Cholecalciferol by formation of its cyclodextrin complex.

Following oral administration to rats of beta-cyclodextrin inclusion complex of 3H-labelled vitamin D3 (cholecalciferol) resulted in significantly higher blood radioactivity as with the non-complexed vitamin. Difference in the first 90 min was 2,3-2,8 fold, and it remained significantly higher up to the 6th h. After 24 h there was no difference between the blood radioactivity of animals treated with complexed and with non-complexed vitamin D3.

Influencing of resorption and side-effects of salicylic acid by complexing with beta-cyclodextrin.

After oral administration of 14C-labelled salicylic acid and its beta-cyclodextrin complex to rats, the blood radioactivity-level increases in the first 2 h than decreases. The blood level obtained with the inclusion complex is somewhat but not significantly lower than with free acid. Since the resorption of cyclodextrin is a considerably slower process, it is very likely that the resorption of salicylic acid take place in the form of free acid after dissociation of the complex. The urinary excretion cumulative curves show that the free salicylic acid is completely excreted, while about 10% of the salicylic acid administered in the form of complex is lost. The cyclodextrin complex formation increases the pK value of all hydroxy-benzoic acids. Direct observations reveals that complex formation decreases the stomach-irritating effect of salicylic acid. The ratio of radioactivity was nearly the same in the organs of animals treated by both free salicylic and cyclodextrin complex.

Intestinal absorption of 14C-labelled beta-cyclodextrin in rats.

beta-Cyclodextrin and glucose labelled universally by 14C was administered orally to rats and blood radioactivity level was followed. In the case of glucose about 20% of the administered radioactivity, related to 10 ml blood was to be found and this appeared in the blood within 10 min following the oral administration. In the case of beta-cyclodextrin maximum 5% of the administered activity could be found in blood, and even this only 4 to 10 h following administration. Presumably, beta-cyclodextrin cannot be absorbed either from the stomach or from the small intestine, only the labelled open-chain dextrins and glucose formed from cyclodextrins on amylase action. Therefore it is very likely that the orally administered beta-cyclodextrin cannot provoke toxic symptoms.

Transport parameters and stoichiometry of active calcium ion extrusion in intact human red cells.

Ca2+-transport and its energy consumption were studied in intact human red cells loaded with Ca2+ by the aid of the ionophore A23187. After the complete elimination of the ionophore the passive Ca2+-permeability of the membrane returned to its normal low value, except when the intracellular Ca2+-concentration was higher than 3 mM or the ATP level fell below 100 muM. Within these limits the rate of Ca2+-extrusion was independent of the cellular ATP content but was greatly enhanced by increasing [Ca2+]i and reached a plateau at about 1 mM intracellular Ca2+-concentration. The maximum rate of Ca2+-efflux was about 85 mumol/l of cells per min at 37 degrees C, pH 7.4. The activation energy of active Ca2+-extrusion was found to be 15 200 cal/mol, and the optimum pH in the suspension was 7.7. Ca2+-efflux was not connected with the counter-transport of cations. The Ca2+-pump was not affected by ouabain or oligomycin and only partial inhibition could be achieved by the SH-reagents: ethacrynic acid, N-ethylmaleimide and p-chloromercuribenzoate or with propranolol and ruthenium red. An 80 to 95% inhibition of the active Ca2+-extrusion was brought about by 50-250 muM lanthanum, which in the above concentrations caused no aggregation or haemolysis. The inhibition of the Ca2+-pump by lanthanum was found to be reversible, the site of inhibition being at the external surface of the cell membrane. To examine the energy consumption of the Ca2+-extrusion, ATPase activity was assessed by measuring inorganic phosphate liberation in Ca2+-loaded red cells the metabolism of which was inhibited by iodoacetamide + Na+-tetrathionate. Ca2+-activated ATPase activity connected with the Ca2+-pump was distinguished from other Ca2+-ATPases by using the non-penetrating inhibitor, lanthanum. The molar ratio of Ca2+-transported per ATP split was found to be 2 : 1.