Toxicology and Hydroxypropyl B Cyclodextrin
Acute toxicity testing has not determined an LD(50) for HPBCD. 5000mg/kg have been administered orally to rats and no mortality was observed. (1)
C14 labeled HPBCD has bee administered orally to determine the metabolic fate of the HPBCD (2,3) . Most of the label was excreted in the feces. 3-6% of the label was absorbed and some label appeared in the blood about five minutes after administration indicating some absorption from the stomach. About 3% of the label appeared in the urine and another 3.25% in the exhaled CO2. The HPBCD preparation did contain some propylene glycol that was also labeled. The amount of label absorbed corresponded to the amount of proplyene glycol present (2)
Teratogenicty and embryotoxicity studies have been done in rats and rabbits at doses up to 5000 mg/Kg per day in rats and 1000mg/Kg per day in rabbits (1). No maternal toxicity, embryotoxicity or teratogenicity was found in rats. In rabbits, no teratatogenicity was observed, but slight maternal and embryotoxicity was observed at 1000 mg/Kg.
Chronic studies were done using both mice and rats. The study with mice was terminated after 104 weeks (4). The mice received 500mg/Kg HPBCD. No adverse histopathology was noted that could be attributed to HPBCD and the mice given HPBCD had a lower incidence of tumors than the controls. A two-year study was also done with rats with a dose of 500 mg/Kg per day (5) . No effects were observed that were attributed to HPBCD.
In an acute study with cynomologus monkeys, a dose of 10, 000 mg/Kg was not lethal (6)
HPBCD is quickly cleared after administration. After a single intravenous administration of (14)C labeled HPBCD, a half-life in the plasma of 0.4 hours and 0.8 hours was found for rats and dogs respectively (3). A plasma clearance of 512 and 188 ml/kg/h was calculated for rats and dogs.
The subchronic toxicity studies with rats (3), no adverse effects were found in rats treated intravenously with 50 mg/Kg HPBCD. At 100 mg/Kg HPBCD. At 100 mg/Kg some minimal histological changes were found in the epithelial cells of the urinary bladder, kidney tubular cells and in the liver. At 400 mg/Kg,there was a decreased body weight and food consumption, increased water consumption,decreased hematocrit, hemoglobin and erythrocyte levels, increased creatin- ine, total bilirubin and aspartate and alanine aminotransferase levels. Some organ weights also increase. Most of these changes were reversible after one month except for slightly elevated aspartate and alanine aminotransferase levels and histological changes in the lung and urinary tract that were only partially reversible.
In subchronic toxicity studies, no adverse effects were found in rats treated intravenously with 50mg/Kg or in dogs receiving 100 mg/KG HPBCD(1). At 400 mg/Kg in dogs therewere slight increases in serum alanine and aspartate aminotransferase and total bilirubin. Histological changes werefound in the lung and epithelial cells of the urinary bladder and renal pelvis. All of the changes were reversed within a month after treatment except for incomplete re- versibility of the swollen renal plevis epithelium.
Teratogenicity and embryotoxicity studies have been done in rats and rabbits at doses up to 400mg/Kg per day(1). Slight maternal toxicity was observed in rats at 400mg/Kg but there were no primary adverse effects in the offspring. No adverse effects were observed in the rabbits.
Dermal irritation studies were done with albino rabbits(7). No erythema, edema or other dermal effects were observed and the material was considered to be a nonirritant to the skin. A dermal sensitization study was done using guinea pigs(8). No irritation was detected upon the initial intradermal injection of HPBCD. Upon challenge of applying HPBCD to the surface of the skin, low incidences of very faint erythema were found in 30% of the animals at 48 hours and 10% of the animals at 72 hours.
HPBCD is nonmutagenic. Mutagenicity was tested using S. typhimurium and E. coli WP2 strains both with and without microsomal activation and found to be non-mutagenic(9). Testing with mammalian cell culture, with and without microsomal activation, also found HPBCD to be non-mutagenic(10).
A limit test using a single concentration was performed using Daphnia magna. Both the EC(50) and no-effect observed concentration (NOEC) are greater than 1084mg/L(11).
2. Fish-a limit test using a single concnetration was performed using Zebrafish (Brachydario rerio). Both the Ec50 and NOEC are greater than 1131mg/L. (12)
3. Algae-a limit test using a single concentration was perfromed using Selenastrum capricornutum. Both the EC50 and NOEC are greater than 1153mg/l.
Cyclodextrin inclusion is a molecular phenomenon in which usually only one molecule of guest interacts with the cavity of the cyclodextrin to become entrapped, unlike other encapsulation methods in which more than one molecule of guest is entrapped in the encapsulation matrix. In order to form a complex with HPBCD to form a stable assocication. A variety of non-covalent forces, such as van der Waal forces, hydrophobic interaction, dipole moment and other forces are responsible for formation of a stable complex.
In most cases, only one colecule is included in the cavity of HPBCD. In the case of some low molecular weight guests, more than one molecule of guest might fit into the cavity. In the case of some high molecular weight molecules, more than one molecule of cyclodextrin might bind to the guest. Only a portion of the molecule must fit into the cavity to form a complex. As a result, a one-to-one molar ratio is not always achieved, especially with high or low molecular weight guests and some prelimiary complex formation and analysis is needed to determine relative amounts of cyclodextrin and guest to be added for complexation.
Hydroxypropyl B-cyclodextrin is very soluble in water and is used to solubilize guest molecules (14). The guest associates with the cyclodextrin so that the hydrophobic portion of the guest interacts with the hydrophobic cavity of the cyclodextrin. This interaction is an equilibrium reaction.
[Guest] + [HPBCD] -----[Guest-HPBCD]
The direction of the equilibrium is dependent upon the guest. For some guests, the complex is predominant while for other guests, the free state might be preferred. In order to reduce the probability of free guest self-associating to form an insoluble precipitate, excess HPBCD is frequently used to increase the probability of the guest existing in the cavity of cyclodextrin and thus to be solubilized rather than associating with other molecules of the guest to form an insoluble mass.
Water is the perferred solvent for complexation. The quest, or portion of the quest which complexes with the cavity of the cyclodextrin is non-polar and perfers the non-polar environment of the cavity of the cyclodextrin rather than the polar aqueous environment. As a result, water provides a driving force for the complexation reaction in addition to dissolving or dispersing the cyclodextrin and guest.
Because of the high solubility of the hydroxypropyl beta cyclodextrin, complexes are also very soluble. Generally the solubility of the complex is not exceeded. If the solubility of the complex is exceeded, the precipitate is amorphous and has a tendency to remain suspended rather than to quickly settle. Addition of a small amount of water will dissolve the precipitate to give a clear solution.
Hydroxypropyl B-cyclodextrin is very soluble in water and heat is not needed to increase the solubility of the hydroxypropyl beta cyclodextrin. At very high concentration of hydroxypropyl B-cyclodextrin (60% and greater), increased temperature can be used to reduce the viscosity of the solution. Increased temperature can also be used to increase the solubility of the guest to increase the rate of complexation.
Heat can destabilize complexes and the temperature at which a complex is stabilized is dependent upon the guest.Temperature and holding time for complexation must be optimized for each guest.
Water is the preferred solvent for complexation. Water is very polar and the guest or portion of the guest which complexes with the cavity of the cyclodextrin is apolar and associates much more readily with the apolar cavity of the cyclodextrin than with water. As a result, the water provides a driving force for complexation.
Not all guests are sufficiently soluble in water making complexation either very slow or immpossible. Complete solubilization of the guest is not necessary. A small amount of guest must be soluble to form a complex. As the soluble guest is completed, more of the guest will dissolve to form more complex. Small amounts of water miscible solvents can be used to assist in dissolution of guests to make the complexation reaction proceed more rapidly. A polar solvent, such as acetone or diethyl ether, which does not complex with the cavity, is preferred in order to minimize competition for the cavity with the guest. Water miscible solvents can be added to the water to dissolve the guest, or the guest can be dissolved in a small amount of solvent and the solution added to the hydroxypropyl B-cyclodextrin solution.Upon addition of the dissolved guest to the solution of cyclodextrin, the guest may be solubilized or dispersed as a fine precipitate. In the latter case, a long stirring or complexation time might be needed, bu complexation occurs more rapidly than if the guest were still in the form of large crystals. Excessive amounts of solvent reduce the driving force for complexation by reducing the difference in polarity between the cavity of the cyclcodextrin and the bulk solution, which can result in good solubilization of the guest, but little or no complexation. Vacuum evaporation of the solvent might result in complexation occuring as the solvent is removed.
Because HPBCD is made up of a mixture of molecular species, there is no direct assay for HPBCD. Instead of assaying for HPBCD directly, assay of attributes and expected contaminants is performed.
Degree of Substitution is the average number of hydroxypropoxy groups per cyclodextrin. Some individual molecules in the mixture will have less than the average and some will have more substitutions than the average. The degree of substitution can be determined by TIR (Fourier Transform InfraRed Spectroscopy) or NMR (Nuclear Magnetic Resonance Spectroscopy). Molecular weight is calculated determining the degree of substitution. Molecular weight is equal to the molecular weight of BCD plus the product of the molecular weight of the hyddroxypropoxy group times the degree of substitution.
MW = 1135 + (58)(DS)
Specific rotation can be used as a measure of purity. The specific rotation is dependent upon the degree of substitution and decreases as the degree of substitution increases.
The expected contaminants in HPBCD result from the synthesis. These are salt and propylene glycol.
The load or amount of guest in a complex is in many cases is determined by measurement of the amount of materials added or by chemical analysis. For chemical analysis, the complex is usually dissolved. The amount of guest is determined by conventional means used for assay of the guest, such as light absorption or chromatography. For spectroscopic determination of the guest, appropriate controls should be used since complexation with HPBCD can cause the light absorption of some compounds to change by altering the ^max or intensity of absorption of light to change. The guest can also be extracted from an aqueous solution of the complex into an organic solvent and assayed using HPLC. Extraction is done at 60'C since the high temperature will assist in disrupting the complex. The amount of HPBCD can be determined using a total carbohydrate assay, such as the DuBois assay. Appropriate controls should also be used for these assays since the guest can effect the results.
Advantages compare to BCD
The main advantage of HPBCD is the increased solubility of the complexes compared to BCD. For most guest, the increase is somewhere between 10 and 100.
The ability of cyclodextrins to stabilize guest is retained in HPBCD. Due to the substitution, binding can be altered which can cause differences in the amount of stability obtained compared to BCD.
HPBCD is listed in TOSCA. The FDA has approved two pharmaceutical formulations that contain HPBCD. One is for oral use and the other for i.v. use. There is a USP monograph in the draft status. ELINCS registration has proceeded through the base set submission and Level 1 submission is in progress.
HPBCD is used for solubilization and stabilization of guests. By complexing with HPBCD, the guest interacts with cavity of the HPBCD to become entrapped. The outer surface of the HPBCD is very hydrophilic and interacts well with water to carry the guest into solution. Some examples are described below.
Itraconazole is used to treat fungal infections and is insoluble in water. It can be solubilized with HPBCD(15). Co-solvents have been used to solubilize itraconazole for oral dosage, but have not been completely successful because of irreversible precipitation which takes place in the stomach. Use of HPBCD prevents the precipitation from occuring. Itraconazole has a high molecular weight (some-where around 700). Propylene glycol is used as a co-solvent for dissolution of the itraconazole. It is compartible with the HPBCD.
Tolnafate is an antifungal drug that is not very soluble in water. HPBCD was used with a 8% or 3% substitution. Solubility increased 30 fold compared to solubilization with BCD(16). Solubility was greater using the lower degree of substitution. The stability constant for the lower degree of substitution was 1109 M-1, 1460 M-1 for the higher degree of substitution and 1860 M-1 for BCD. The complex in HPBCD had a faster dissolution rate than the BCD complex.
Some proteins can also be solubilized with HPBCD(17). Ovine growth hormone is a protein with a molecular weight of 20,000 and is not soluble in water at physiological pH. In water it is not solubilized until pH 11.5. Using HPBCD, the hormone could be solubilized at pH7.5 to pH8.5.
1. W. Cousssement,H. Van Cauteren,J. Vandenberghe, P.Vanparys,G. Teuns,A. Lampo and R. Marsboom (1990). Toxicological profile of hydroxypropyl B-cyclodextrin (HP B-CD) in laboratory animals. In D. Duchene (ed.) Minutes 5th International Symposium on Cyclodextrins. Editions de Sante pp. 552-524.'
2. A. Gerloczy,S.Amtal,I.Szatmari,R. Muller-Horvath and J.Szejtli (1990). Absorption, distribution and excretion of C-labelled hydroxypropyl B-cyclodextrin in rats following oral administration. In. In D.Duchene (ed.), Minutes 5th International Symposium on Cyclodextrins. Editions de Sante pp.507.513.
3. J. Monbaliu, L.van Beijsterveldt, W. Meuldermans, S. Szathmary, and J. Heykants (1990).Disposition of hydroxy- ropyl B-cyclodextrin in experimental animals. In D.Duchene (ed.).minutes 5th International Symposium on Cyclodextrins. Editions de Sante pp. 514-517.
4. J.J. Wallery, M.E.Shaw, J.T. Yarrington, M.S.Werley,T. M. Sullivan and A.W. Singer (1997). Effects of cage type, food type,and hydroxypropyl-B-cyclodrextrin on chronic toxicity/oral carcinogenicity study parameters in CD-1 mice. Bttelle. Poster presented at 1997 Annual SOT Meeting.
5. M.E. Shaw, D.M. Sells,T. Sullivan and A Singer (1997). Battelle,Poster presented at the 1997 Annual SOT Meeting. 6. M.E. Brewster.,K.S. Estes and N. Bodor (1990). An intravempis toxicity study of 2-hydroxypropyl-B-cyclodextrin,a useful drug sollubilizer in rats and monkeys. International Journal of Pharmaceutics 59: 231-243.7.
7.Primary Dermal Irritation Study in Albino Rabbits with Hydroxypropyl Beta Cyclodextrin (1993). Study report
8.Dermal Sensitization Study (Maximization Design in Guinea Pigs) with Hydroxypropyl Beta Cyclodextrin (1993). Study report
9-13 (research reports)
14 Solubilization of pharmaceuticals by HPCD is covered by patents such as US 4,727,064 "Pharmaceutical preparations containing cyclodextrin derivatives" and EP 0 149 197 "Pharmaceutical compositions containing drugs which are sparingly soluble in water or instable and methods for their preparations" and others.
15 US Patent 5, 707,975.
16 D. Peri, C.M. Wandt, R.W. Cleary, A.H. Hikal and A.B.Jones (1994). Complexes of tolnaftate with beta-cyclodextrin and hydroxypropyl beta-cyclodextrin. Drug Development and Industrial Pharmacy 20:1401-1410.