Enhanced intestinal absorption of BCS class IV
drug Acetazolamide using nano-hydroxyapatite
and investigation of the mechanism involved

P-272○ Ryosuke Miyasaka, Shogo Komiyama, Keiichiro Kikukawa, Roslyn Hayman
Sangi Co., Ltd, Central Research Laboratory

PurposeIn recent years, poorly water-soluble and poorly absorbable drugs are increasing in number, so to facilitate their use in oral
formulations, techniques for improving such physical properties are required. We are attempting to improve the solubility andintestinal absorption of such drugs using nano-hydroxyapatite (n-HAP). Acetazolamide (AZ), belonging to BCS class IV,
was used as one such model drug. An AZ/HAP formulation was prepared by coating the surface of AZ with n-HAP
mechanically. SEM observation, a solubility test and a pharmacokinetics study were performed compairing AZ and AZ/HAPformulation. We further investigated the localization of n-HAP in the small intestine and major organs histologically using
fluorescent dye to assess whether, after ingestion, the n-HAP might be absorbed into the body together with the drug.

SEM observation

Concentration of Acetazolamide
dissolved (μg/ml)

Solubility test (n=3)
Respectively, 0.3 g of AZ and 0.9 gof AZ/HAP formulation (containing0.3 g of AZ) were added to fifty
milliliters of dissolution test solution 2(pH 6.8) and stirred at a constantrotation. One milliliter samples werecollected at 3, 10, 30, 120 and 360 min.
Each sample was centrifuged for 3 min
at 12,000 rpm, and the concentration ofAZ in supernatant was determined byspectrophotometer.

3000
2500
2000
1500
1000
500
0060120(A)180240300

360Time (min)●：AZ■：AZ/HAP formulationThe mean amount of AZ dissolution
from AZ/HAP formulation wassignificantly higher than that of AZ atall points, but it was not greatlydifferent.

Blood concentration ofAcetazolamide (μg/ml)

Pharmacokintics study (n=4)(B)(A) AZ crystals, (B) AZ/HAP formulation

The major axis of AZ was about 20 µm. The size ofAZ/HAP formulation was almost the same as that of AZ
and the surface of AZ in the formulation was uniformly
coated with n-HAP particles.

181614121086420AZAZ/HAP06AUC(0-6)(μg/ml・h)
10.52±1.87
37.74±3.64

AZ and AZ/HAP formulation wereorally administered to Kwl:SD malerats (8 weeks, 10 mg/kg AZ). A blood
sample was collected at 0.5, 1.0, 3.0,6.0 and 24.0 hr after administration.
The blood concentration of AZ wasdetermined by HPLC.

18

The AUC (0-6) value in the case ofAZ/HAP formulation was about 3.6
times that for AZ. It was thought thatfactor other than solubility contributed
to the improvement of intestinal
absorption in AZ/HAP formulation
because the solubility of AZ/HAP
formulation was not greatly differentfrom that of AZ.

1224

Time (hr)●：AZ■：AZ/HAP formulation

Localization of n-HAP in the intestine and liver
Merge(A)OsteoSense 680EX

Merge

OsteoSense 680EX

Merge

OsteoSense 680EX

CalseinCalseinCalsein

DAPI

DAPI

DAPI

(B)

(C)

(A) Villi, (B) Peyer’s patch, (c) Liver (enlargement of the area surrounded by dotted lines also shown)

n-HAP particles were doubly labeled with fluorescent dyes Calcein and OsteoSense 680EX. Fluorescent dye-bound n-HAP was orally administered toKwl:SD male rats (8 weeks, 300 mg/kg). Rats were euthanized at 30 min after the administration, and the intestine and liver were excised immediately.
Tissue frozen sections were prepared and nuclear staining done with DAPI. The localization of n-HAP in the tissue frozen sections was examined with
confocal laser scanning microscope.
n-HAP particles were observed in the intestinal villi, Peyer’s patch and liver (shown by arrows), indicating that the n-HAP particles were absorbed into
the body via the intestinal villi and Peyer’s patch, quickly reaching the liver. This strongly suggests that AZ bound to n-HAP in the intestine, may beabsorbed into the body together with n-HAP.

Conclusions

Contact address

The intestinal absorption of AZ, a poorly water-soluble and poorly
absorbable drug was improved by coating the surface of the drug withn-HAP particles, although solubility was not greatly improved. n-HAP
particles were absorbed into the body via the intestinal tract within 30 min
after administration, quickly reaching the liver. These results suggest that
AZ is absorbed into the body together with n-HAP and that the n-HAP
coating may be a major factor in the improvement of intestinal absorption
shown by the AZ/HAP formulation.

Sangi Co., Ltd,Ryosuke Miyasaka
E-mail:miyasaka@sangi-j.co.jp
URL:http://www.sangi-co.com

COI disclosure
We have no conflict of interest with regard
to this presentation.