Molecular Specificity of Cyclodextrin Complexation - ACS Symposium

Chapter 19

Molecular Specificity of Cyclodextrin Complexation 1

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Ching-jer Chang , Hee-Sook Choi , Yu-Chien Wei , Vivien Mak , Adelbert M. Knevel , Kathryn M. Madden , Gary P. Carlson , David M. Grant , Luis Diaz , and Frederick G. Morin 1

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Downloaded by CORNELL UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0458.ch019

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Department of Medicinal Chemistry and Pharmacognosy and Department of Pharmacology and Toxicology, School of Pharmacy and Pharmacal Sciences, Purdue University, West Lafayette, IN 47907 Department of Chemistry, University of Utah, Salt Lake City, UT 84112 3

The mostremarkablemolecular feature of cyclodextrins is their ability to form inclusion complexes with numerous guest molecules without a covalent bond being formed. Molecular specificity of cyclodextrin inclusion complexation was elucidated by determining the dynamics of molecular motion and relaxation, and local electricfieldgradient in the solid state as well as by analyzing the chemical shifts and couplings of high resolution NMR, and fast atom bombardment mass spectral data. The molecular specificities with benzaldehyde and tolbutamide provided fundamental information for understanding the molecular mechanisms involved in the enhancement of in vitro antitumor activity of benzaldehyde in human tumor cell lines and in vivo hypoglycemic effects of tolbutamide in rabbits. It is widely recognized that regiospecificity and stereospecificity of most chemical and biochemical reaction specificities are governed by a prior "complexation" process (host-guest or intermolecular recognition). In recent years, our understanding of the molecular specificity in the intermolecular recognition has been greatly enhanced by many newly developed physical and chemical methods. In particular, NMR spectroscopy has developed into a perpetually expanding and excitingresearchmethod by theremarkableimprovements in both hardware and software designs which take advantage of the versatile computer capabilities and highfieldsuperconducting technology. Studies on the intermolecular complexations in solution have, however, often encountered some intrinsic difficulties. Firstly, the complexation processes in solution can be highly complicated due to dynamic chemical exchanges, solvent interference and conformational fluctuation. Secondly, the solubility of complexes may be limited which greatly reduces the sensitivity of detection. Thirdly, chemical decomposition and rearrangement may occur in solution Most of these problems can be circumvented/simplified by studying the complexation process in the solid state. This restricts stringently the intramolecular motion and intermolecular exchange, and simultaneously overcome solubility and stability problems. Therefore, one of the primary methods employed by us in investigating the molecular specificity of cyclodextrin complexation was solid state carbon-13 nuclear magnetic resonance (^CNMR) spectroscopy using the combined techniques of high power proton decoupling, magic angle spinning (MAS) and carbon-hydrogen crosspolarization (CP) (1,2). 0097-6156/91/0458-0296$06.25/0 © 1991 American Chemical Society

In Biotechnology of Amylodextrin Oligosaccharides; Friedman, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

19. CHANG ET AL

Molecular Specificity of Cyclodextrin Complexation


SOLID STATE C-13 NMR of a-CYCLQDEXTRIN The merits of this new methodology can be clearly illustrated in the ^CNMR analyses of cyclodextrin-water complexes. The solution C-13 spectrum of α-cyclodextrin shows only six signals for the six carbon resonances of the glucose units. This indicates the rapid motion of α-cyclodextrin and fast exchange of water molecules. Thus it provides a non-specific and time-averaged spectrum. However, this motion and exchange are largely restricted in the solid state. The solid state CP/MAS C-13 spectrum of α-cyclodextrin (recrystallized from water) distinctly displays resolved C-l and C-4 resonances from different oc-(l — > 4)-linked glucose units in the macrocycle, indicative of the molecular asymmetry and specificity of 56 KHz) resulted in the same spectral feature, suggesting that dipoledipole coupling itself may not be the direct cause. An alternative explanation was suggested by Inoue et al. (14) based on the earlier studies of Rothwell and Waugh (15). The C-13 resonances could be greatly broadened by inefficient decoupling because the motion frequency of the aromatic ring is close to the nutation frequency of the proton decoupling field. X-ray crystallographic data clearly indicate that p-nitrophenol cannot undergo 180° rotation within the cavity (3). Thus, the flip motion must take place within a small range between two C-3 protons or C-5 protons of the neighboring glucoses. This observation has profound implications in the studies of host-guest (enzymesubstrate/inhibitor) recognition by solid state C-13 NMR. Further substantiation of this implication was therefore conducted. The most direct approach to perturb the dynamic efect


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BIOTECHNOLOGY OF AMYLODEXTRIN OUGOSACCHARIDES


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Figure 1. Solid state CP/MAS 25 MHz CNMR spectra of (A) a-cyclodextrin, recrystallized from water and, (B) dehydrated α-cyclodextrin, 50°C under vacuum for 10 nr.


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A

1)0 ' IÉO ' IÉO ' 110

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Figure 2. Solid state CP/MAS 25 MHz CNMR spectra of (A) a-cyclodextrinp-nitrophenol inclusion complex I and, (B) p-nitrophenol, recrystallized from water.


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BIOTECHNOLOGY OF AMYLODEXTRIN OLIGOSACCHARIDES

of molecular motion is to freeze the freedom of motioa Indeed, the protonated carbon signals were recovered by conducting the NMR experiment at -120° (Figure 3). LOCAL ELECTRONIC DENSITY A second a-cyclodextrm-p-nitrophenol 1:1 powdery complex was also obtained during the preparation of the crystalline inclusion complex. Its solution C-13 NMR spectra were identical. However, the solid state C-13 NMR spectrum (Figure 4) appears remarkedly different Most of the protonated carbon resonance signals are still retained whereas the nitroattached carbon is changed from a doublet to a singlet The initial splitting of this carbon resonance is due to the C-13 and N-14 quadrupole coupling which cannot be fully reduced in the magic angle spinning mode at the magnetic field strength of 2.35T (25MHz). The magnitude of this splitting is inversely proportional to the ratio (Z/A) of the Zeeman frequency (Z) to the quadrupole coupling constant (A=e Qq/h) (16). Since the spectra of p-nitrophenol and its complex are measured at constantfield,the Zeeman frequency remains unchanged. Then the reduction of this quadrupole splitting is most likely ascribed to the reduction of the quadrupole coupling constant. This surprising finding indicates the decrease of the local electricfieldgradient (q) after the inclusion complex with α-cyclodextrin is formed because all other factors (e: electronic charge, Q: N-14 electric quadrupole moment, and h: Planck's constant) involved in the quadrupole coupling constant are invariable. This directly manifests that the perturbation of the local electricfieldgradient in the non-covalent host-guest complex can be detected by a direct NMR method. One of the possible complexation probabilities is that the second complex may be a non-specific adsorbed complex formed primarily by intermolecular hydrogen-bonding. An independent procedure was designed for preparing the adsorbed complex by mixing a methanol solution of p-nitrophenol directly with α-cyclodextrin and then quickly removing methanol under reduced pressure. The solid state C-13 NMR spectrum of the resulting adsorbed complex (Figure 5A) looks somewhat similar to that of the second complex. However, p-nitrophenol in this adsorbed complex is completely removed by washing with cold ether or methylene chloride (Figure 5B) since p-nitrophenol is not included in the cavity. A similar washing process failed to remove p-nitrophenol from the second complex, strongly suggesting that the second complex is very likely an inclusion complex. The most plausible structure for the second inclusion complex is that, in contrast to the first inclusion complex, the nitro group of p-nitrophenol is oriented toward the outside of the α-cyclodextrin cavity.


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MOLECULAR RELAXATION The degree of free motion of the substrate in the active site is critical in determining the stereospecificity of a biochemical reaction. It is thus important to study the freedom of motion of the guest molecule in the inclusion complex. Adamantane has been shown to possess plastic crystal structures with high freedom of motion in the solid state. Thus, its carbon signals in the solid state are extremely sharp (Wi/2 < 1Hz) (12). The solid state spectrum of the β-cyclodextrin-l-adamantanecarboxylic acid complex revealed the asymmetric structure of the dimeric carboxylic acid (IS) in the inclusion complex (Figure 6A). A unique way for quantitatively assessing the freedom of motion is to measure the relative rate of losing carbon magnetization after the C-13/H-1 spin-locked process is terminated. This dipolar dephasing experiment is performed by turning off the proton decoupler for a short period prior to the acquisition of carbon signals (12). The dipolar dephasing spectrum of the complex shows that all the cyclodextrin peaks are almost diminished whereas all the l-adamantenecarboxylic acid signals are still retained (Figure 6B). This result indicates that the guest molecule in the cavity of β-cyclodextrin has much greater freedom of motion than that of the host molecule. This technique provides a new approach to monitor selectively the variations of guest or substrate/inhibitor in the biochemical intermolecular complexes.


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Figure 3. Solid state CP/MAS 50 MHz CNMR spectra of a-cyclodextrin-pnitrophenol inclusion complex I at (A) room temperature and, (B) -120°C.


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A

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Ί Ι ο ' -150-

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130 110

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Figure 4. Solid state CP/MAS 25 MHz CNMR spectra of (A) a-cyclodextrinp-nitrophenol inclusion complex Π and, (B) p-nitrophenol, recrystallized from water.


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Figure 5. Solid state CP/MAS 25MHz CNMR spectra of (A) a-cyclodextrinp-nitrophenol adsorbed complex and, (B) after washed with cold ether.




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Figure 6. Solid state CP/MAS 25 MHz C N M R spectra of β-cyclodextrin-ladamantane carboxylic acid inclusion complex; (A) Regular acquisition, (B) Dipolar dephasing acquisition (dephasing time: 40 usee).


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MOLECULAR ENCAPSULATION AND BIOAVAILABILITY Each guest molecule in a cyclodextrin inclusion complex is encapsulated by cyclodextrin. This molecular encapsulation can profoundly modify the chemical and physical properties of the guest molecules. Its potential applications have been shown in (1) stabilization of air- or light sensitive substances, (2) enhancement of water solubility, (3) suppression of unpleasant taste or odor and (4) improvement of bioavailability (20.21). Our primary interest focuses on elucidating the structure specificity in cyclodextrin complexation and thereby providing a definite basis for understanding their molecular mechanisms.


INCLUSION COMPLEXATION OF BENZALDEHYDE Previous interest in the antitumor activity offig(Ficus carica L.) fruit led to the isolation of benzaldehyde as a major active component (22). Its carcinostatic effect was attributed to selective inhibition of the uptake of nucleosides and carbohydrates, and the reduction of intracellular adenosine 5'-triphosphate level (22). However, benzaldehyde is an oilly liquid and only sparingly water soluble. Its instability in air and light presented considerable problems in the delivery and formulatioa Takeuchi et al. (24)firstprepared the complex with α-, β- and γ-cyclodextrin. More importantly, the x-ray structure of a-cyclodextrinbenzaldehyde 1:1 complex was subsequently determined (25) , providing a working basis for uncovering its structure in solution. 1

NMR ANALYSIS: The 470 MHz HNMR spectra of benzaldehyde before and after the formation of α-cyclcdextrin displayed afirst-orderpattern. The results are summarized in Table I. Spectra of α-cyclodextrin before and after inclusion of benzaldehyde clearly showed a second-order pattern (Figure 7). Arigorouscomputer spin-simulation allowed us to determine their chemical shifts and coupling constants (Tables I and Π). The calculated values appear in reasonable agreement with those reported by Wood et al. (26) except for the H-6' protons. The discrepancy which appears is presumably due to the limited resolution of the previously measured 220 MHz ^HNMR spectrum. A 500 MHz spectrum of α-cyclodextrin recently reported by Yamamoto and Inoue (27) is almost identical to our spectrum. However, no attempt was reported to calculate the accurate chemical shifts and coupling constants.

INCLUSION STRUCTURES: The single-crystal x-ray analysis of the a-cyclodextrinbenzaldehyde (1:1) complex (25) indicates that the phenyl ring of benzaldehyde is the leading group inserted into the center of the α-cyclodextrin cavity from the broader end and the aldehyde group protrudes from the cavity (structure A). However, the ortho proton chemical shift change (Δδ=32.4 Hz) is larger than the para (Δδ=21.6 Hz) or meta (Δδ=21.2 Hz) proton chemical shift change upon formation of α-cyclodextrin inclusion complex (Table I). This suggests that in solution the aldehyde group is the leading group included into the cavity

(structure B). This tentative structure is corroborated by the solution ^CNMR results. The upfield shift for the aldehyde carbon (-23.8 Hz) and C-l (-16.1 Hz) resonances is a good indication for the changes of dielectric environment from the high dielectric water medium to the low dielectric cavity of cyclodextrin. Furthermore, the nuclear Overhauser effect (Figure 8) between the aldehyde proton and the H-5' protron of α-cyclodextrin strongly favors structure Β for the a-cyclodextrin-benzaldehyde inclusion complex in solution.


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154.0

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Figure 7. 470 MHz HNMR spectra of α-cyclodextrin-benzaldehyde inclusion complex in pD 7.4 phosphate buffer solution . (A) measured, (B) calculated based on Raccoon spin simulation program.


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307 Molecular Specificity of Cyclodextrin Complexation

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Table I.

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470 MHz HNMR chemical shift of benzaldehyde before and after complexation with α-cyclodextrin in pD 7.4 phosphate buffer solution (DMSO-d6 was used as an external reference)


CHO

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Protons CHO 2,6 3,5 4 r 2' 3' 4' 5' 6'a 6'b

Table Π.

a-Cyclodextrin benzaldehyde 1:1 a-Cyclodextrin Complex (ppm) (ppm) 9.9680 8.0280 7.7670 7.8060 5.0330 3.6080 3.8880 3.5703 3.8110 3.9000 3.8535

Benzaldehyde (ppm)

Difference (Hz) 15.5 32.4 21.2 21.6 - 8.5 -10.3 -44.2 - 6.0 -12.2 - 3.3 - 4.9

9.9350 7.9590 7.6250 7.7600 5.0510 3.6300 3.9820 3.5830 3.8370 3.9070 3.8640

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470 MHz HNMR coupling constants (Hz) of α-cyclodextrin and acyclodextrin-benzaldehyde complex in pD 7.4 phosphate buffer solution

Coupling

a-Cydodextrin

Jl2 Jl5 J23 J34 J45 J46a J46b J56a J56b J6a6b

3.4 -0.7 10.1 9.2 9.5 -0.6 -0.5 2.0 4.4 -11.5

a-Cyclodextrin-Benzaldehyde 3.5 -0.5 9.8 9.2 9.4 -0.7 -0.7 1.8 4.3 -12.5




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Figure 8. 200 MHz two-dimensional nuclear Overhauser enhancement spectrum of α-cyclodextrin-benzaldehyde complex in pD 7.4 phosphate buffer solution.


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6-OH

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AIJTQXIDATTON: The autoxidation rates of free benzaldehyde and its inclusion complex with α-cyclodextrin in neutral solution at 70°C were measured by HPLC. The half-life for the a-cyclodextrin-benzaldehyde was 6.2 nr., 3.6 times longer than that for benzaldehyde alone (1.7 nr.), indicating that the aldehyde group is included into the cyclodextrin cavity which prevents oxidation by air. ANTITUMOR CYTOTOXICITY: The stabilization effect of benzaldehyde by acyclodextrin is also reflected in the in vitro antitumor cytotoxicity against human tumor cell lines: A-549 (non-small-cell lung carcinoma), MCF-7 (breast adenocarcinoma) and HT-29 (colon adenocarcinoma) (Table III). α-Cyclodextrin-benzaldehyde inclusion complex is about 5-8 fold more cytotoxic than free benzaldehyde.

Table HI. In vitro antitumor cytotoxicity against human tumor cell lines ED50 (μ mole/ml) Compound

A-549 (lung)

MCF-7 (breast)

HT-29 (colon)

Benzaldehyde a-Cyclodextrin a-Cyclodextrin-Benzaldehyde

0.19 0.98 0.04

0.68 0.88 0.08

0.50 0.59 0.06

INCLUSION COMPLEXATION OF TOLBUTAMIDE Tolbutamide [l-butyl-3(p-tolylsulfonyl)urea] is a first-generation hypoglycemic drug used clinically in the treatment for insulin-dependent diabetic patients in whom the pancreas retains the capacity to secrete insulin (2S) . Its poor water solubility and dissolution rate are considered to be the rate-limiting steps in the gastrointestinal absorption. The low water solubility and dissolution rate are likely due to the strong intermolecular hydrogen-bonding


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between the sulfonylurea groups, which is then encircled by two hydrophobic end groups. Modification of tolbutamide dissolution properties by water-soluble polymers has been used to improve oral bioavailability (29-321 Cyclodextrin complexation of tolbutamide was first reported in 1978 to enhance its water solubility (22). The improvement of the bioavailability of tolbutamide by β-cyclodextrin was recently demonstrated by Vila-Jato et al. (24).


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Results of most previous experiments suggested that tolbutamide forms inclusion complexes with β-cyclodextrin in a 1:1 ratio (34-36V However, tolbutamide consists of two hydrophobic end groups (tolyl and butyl) and can potentially form a 2:1 complex which should provide maximum enhancement of water solubility. The complex was prepared by dissolving both β-cyclcdextrin (2 mmoles) and tolbutamide (1 mmole) in pH 11.0, 0.05M phosphate buffer solution (160 ml) at room temperature. Slow precipitation occurred after neutralization to pH 7.0 and then concentration by vacuum evaporation. The precipitate was further recrystallized from water. The *HNMR spectra of die initial precipitate and the recrystallized solid (mp: 268°Q showed a molar ratio of 2:1. FAST ATOM BOMBARDMENT MASS SPECTROMETRY ANALYSIS: Using ditrdotrireitoVditWoeiytrm (3:1) as a matrix, the protonated molecular ion (m/z: 2540) for the recrystallized β-cyclodextrin-tolbutamide complex, together with strong potassium adduct (m/z: 2578) and weak sodium adduct (m/z: 2562) peaks are clearly detected (Figure 9). This is a strong indication of a 2:1 complex which is consistent with the integration data of HNMR. 1

FOURIER TRANSFORM INFRARED SPECTRAL ANALYSIS: The ureido functional group of tolbutamide shows two characteristic IR absorptions (Figure 10), at 1662.4 (carbonyl stretching band) and 1559.6 (NH bending band) cm . This indicates a strong intermolecular hydrogen-bonding as is shown by the x-ray crystallographic data (22). This hydrogen-bonding can be disrupted by the formation of cyclodextrin complex as is revealed by a shift in the carbonyl stretching band and NH bending band to 1701.2 and 1542.1 cm" , respectively. The absorption intensity of the complex is also significantly reduced. It probably results from some part of tolbutamide being bound to cyclodextrin, thereby restricting its freedom of vibration. However, these changes do not exclusively indicate the formation of an "inclusion" complex. A more informative variation should be furnished by the changes of the phenyl or butyl group. However, the IR absorptions of these hydrophobic groups cannot be unambiguously determined due either to the weak absorption or the overlap with the cyclodextrin absorption -1

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NMR ANALYSIS: Inoue et al. recently reported the relative chemical shift and coupling constant assignments of β-cyclodextrin at pD 3 and pD 10 with the aid of two-dimensional 500 MHz *H COSY experiments (38). These data provide initial values for computer spin calculation (Figure 11) although only approximate data based on the first-order analysis were shown. All spectral data were measured in 0.2N NaOD solution because of the limited solubility in neutral solution. Upon complexation with β-cyclodextrin marked spectral changes were observed. The precise chemical shifts and coupling constants calculated from computer spin simulation are summarized in Tables IV and V.


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3-CD+Na Downloaded by CORNELL UNIV on August 2, 2012 | http://pubs.acs.org Publication Date: April 30, 1991 | doi: 10.1021/bk-1991-0458.ch019

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Figure 9. Fast atom bombardment mass spectrum of β-cyclodextrin-tolbutamide 2:1 inclusion complex using dithiothreitol/dithioerythritol (3:1) matrix.




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Figure 10. Fourier transform IR spectra of (A) tolbutamide, (B) β-cyclodextrin, ( Q β-cyclodextrin and tolbutamide mixture (2:1) and, (D) β-cyclodextrin-tolbutamide 2:1 inclusion complex.


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Figure 11. 470 MHz HNMR spectra of β-cyclodextrin in 0.2N NaOD solution (A) calculated based on Raccoon simulation porogram, (B) measured.


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Table IV. 470 MHz HNMR chemical shifts (ppm) of tolbutamide, β-cyclodextrin and β-cyclodextrm-tolbutamide in 0.2N NaOD Solution Protons

β-CyclodextrinTolbutamide (2:1) (ppm)

r


τ3' 4' 5' 6'a 6'b 1 2 3 4 7 δ 9

Table V.

β-Cyclodextrin (ppm)

4.9480 3.5095 3.8470 3.4440 3.7420 3.8172 3.7600 0.8170 1.2180 1.3370 2.9650 7.6595 7.2760 2.3690

Tolbutamide (ppm)

4.9460 3.5040 3.8640 3.4370 3.8060 3.8200 3.7775 0.8030 1.2000 1.3250 2.9560 7.6720 7.3255 2.3590

Chemical shift Differences (Hz) 0.9 2.6 -8.0 3.3 -30.0 -1.3 -8.2 6.6 8.4 5.6 4.2 -5.9 -23.2 4.7

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470 MHz HNMR coupling constants (Hz) of β-cyclodextrin and βcyclodextrin-tolbutamide complex in 0.2N NaOD Solution

Coupling

β-Cyclodextrin

J12 Jl5 J23 J34 J45 J46a J46b J56a J56b J6a6b

3.5 -0.6 9.6 9.3 9.2 -1.0 -1.3 2.0 3.8 -12.0

β-Cyclodextrin-Tolbutamide (2:1) 3.8 -0.7 9.4 9.4 8.7 -0.7 -1.4 2.0 4.4 -11.0

INCLUSION STRUCTURE: The upfield shifts for the H-3' and H-5' of β-cyclodextrin upon the formation of tolbutamide inclusion complex are attributed to the anisotropic shielding of the phenylring.This anisotropic shielding offers a direct approach to elucidate the spatial disposition of the aromatic group in the complex by comparing the relative magnitude of chemical shift changes between H-3' and H-5' (2L32). The larger shift of H-5' (30 Hz), relative to the shift of H-3' (8.0 Hz), suggests that the aromaticringextends from the primary hydroxylrimor from the secondary hydroxylrimbut penetrates deeply into the cavity. In 0.2N NaOD solution, both the ureido group of tolbutamide (pKa: 5.4) (4Q) and the secondary hydroxyl groups of β-cyclodextrin (pKa: 12.2) (?6.41.42) should all be ionized. The electric repulsion between the ureido group and the secondary hydroxyl groups should hinder the tolbutamide entry in β-cyclodextrin past its secondary hydroxylrim.Therefore, it is more likely that tolbutamide enters β-cyclodextrin from the primary hydroxyl rim. The hydrogenbonding between the sulfonyl group and the primary hydroxyl group may further strengthen


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complex formation. Previous NMR studies also suggested this favorable structure (22,25). The chemical shift changes for the aromatic protons induced by cyclodextrin complexation are very different from those observed for other aromatic substrates, such as benzaldehyde. In most cases, the aromatic protons undergo a downfield shift (22). The upfield shift for the ortho-protons (H-7) may be ascribed to steric interference by cyclodextrin of the coplanarity between the sulfonyl group and the aromatic ring. However, this steric effect could not account for the large upfield shift (23.2 Hz) of the mete-protons (H-8). One of the most plausible explanations may be the anisotropic shielding of the ether oxygen (0-4*) of βcyclodextrin. HYPOGLYCEMIC EFFECT: The modulation of the bioavailability of tolbutamide by βcyclodextrin was monitored by measuring its hypoglycemic effect with and without βcyclodextrin in male New Zealand white rabbits. Seven rabbits were used in order to eliminate individual differences. Before administering the drug, rabbits were denied food for 24 hours. Blood samples were taken before administering the drug, 30 minutes after oral administration into the stomach directly, and at 1 hour intervals for eight hours. A Beckman glucose analyzer was used to assay the plasma glucose level. The plasma glucose levels versus time after administration of the drugs are shown in Figure 12. When tolbutamide was given alone, the lowest plasma glucose level was obtained after six to seven hours, while tolbutamide administered in the β-cyclodextrin inclusion complex resulted in the lowest plasma glucose level after four tofivehours. The decrease in plasma glucose was significantly greater when the rabbits were treated with β-cyclodextrin complex than when given the tolbutamide alone. Clearly, β-cyclodextrin can significantly improve the bioavailability of tolbutamide. These results were similar to the previously reported results of Vila-Jato et al. (24) except our dosage was only half the size of theirs.

140— · —

Tolbutamide

— • — Tolbutamide-p-CD

120Blood glucose levels (mg/dl)

100-

80·

60 Time (hour)

Figure 12. Modulation of plasma glucose levels in rabbits after oral administration of tolbutamide and its β-cyclodextrin inclusion complex.


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Acknowledgment This research was supported partially by the Purdue Research Foundation, Purdue University, West Lafayette, Indiana, and partially by Grant GM08521-29 from the Institute of General Medical Sciences of the National Institutes of Health, PHS.


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November 20, 1990 In Biotechnology of Amylodextrin Oligosaccharides; Friedman, R.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.

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