Formulation and evaluation of topical solutions containing itraconazole-phenol eutectic mixture

국문 초록 (Abstract)

이트라코나졸은 진균의 피부 감염에 의한 무좀을 치료하는 약물로, 현재 경구용 제제만이 사용되고 있다. 그러나 이트라코나졸을 경구로 투여할 경우, 생체 이용률이 낮고 그 개인차가 크며, 위장관 장애 부작용이 나타난다. 또한, 무좀이란 질병의 특성 상 감염 부위에만 약물이 고농도로 유지되는 것이 치료에 효과적이므로 경구용 제제 대신 외용제로 적용할 경우에 더 높은 치료 효과를 기대할 수 있다.
그러나 이트라코나졸은 피부 투과성이 낮기 때문에, 외용제로 만드는 데 어려움이 있다. 따라서 이트라코나졸의 녹는점을 낮추기 위해 이트라코나졸과 페놀의 공융 혼합물을 사용하고, 기존의 자료보다 고농도의 약물을 로딩하기 위하여 용액으로 제조하였다.
이 실험의 목적은 이트라코나졸과 페놀의 공융 혼합물을 함유한 외용액의 제조와 피부 투과, 침적 그리고 자극 시험을 이용한 평가이다.
피부 투과 실험은 무모 마우스에서 Franz 확산 셀을 이용하여 진행하였다. 이트라코나졸과 페놀의 공융비, 이트라코나졸과 피부 투과 촉진제, 점도 증강제의 농도를 변화시켜 가며 이트라코나졸의 피부 투과에 미치는 영향을 시험하였다. 이트라코나졸의 농도가 증가함에 따라 이트라코나졸의 투과량과 투과속도가 증가하였으나, 안정성 시험 결과에 따라 이트라코나졸의 농도는 5%로 선택하였다. 또한 피부투과 촉진제로 사용된 올레익 액시드는 처방에 첨가할 경우, 이트라코나졸의 투과속도가 유의적으로 증가하였고, 그 원인으로는 올레익 액시드가 피부 지질층의 상변화 온도를 낮춤으로써 지질 구조의 유동성을 증가시키는 것으로 사료된다. 또한, 처방에서 벤질 알코올의 함량이 증가하면 이트라코나졸 투과에의 지연 시간이 증가하고, HPMC 2910의 함량이 증가하면 외용액의 점도가 증가하고 그에 따라 이트라코나졸의 투과 속도는 유의적으로 감소하였다. 이는 점도가 낮을수록 피부층을 통과하여 수용체 상으로 투과되기 용이하기 때문이라 사료된다. 피부 투과 실험 결과, 최종 처방의 피부 투과량은 32.68 ± 4.39 µg/cm2 이었다.
최종 처방과 거기서 페놀을 제외한 처방으로 Sprague-Dawley 쥐에서 피부 침적 실험을 실시하였다. 피부 침적 실험 결과, 각질층에서 두 처방 간에 유의적인 차이는 나타나지 않아서, (P > 0.05) 이트라코나졸의 침적에는 공융 혼합물이 영향을 미치지 않았음을 확인할 수 있었다. 그러나 각질층을 제외한 나머지 피부 층인 진피와 상피에서는 공융 혼합물을 이용한 처방에서 더 적은 양의 이트라코나졸이 침적된 것을 확인할 수 있었다. 이것은 공융 혼합물의 점도로 인하여 마치 reservoir처럼, 외용액에서의 이트라코나졸 방출이 조절된 것이라 사료된다. 또 시간이 지남에 따라 이트라코나졸의 침적량이 감소한 이유로는, 투과 속도가 대사 속도보다 감소한 것에 따른 결과라 사료된다.
피부 자극 실험의 경우, 뉴질랜드 토끼를 사용하여 진행되었는데, 그 결과 이트라코나졸과 페놀의 공융혼합물을 함유한 포뮬레이션에서 피부에 어떠한 자극도 나타나지 않았다.
따라서 이트라코나졸과 페놀의 공융 혼합물을 이용한 외용액의 경우, 이트라코나졸의 높은 피부 투과량과 피부 속도를 나타냈으며, 실제 적용을 위해서는 병리 부위에 적용하여 치료 효과를 확인하는 보충 실험이 필요하다고 생각된다.

다국어 초록 (Multilingual Abstract)

Itraconazole has been used to treat dermatophytosis, mycosis infections of the skin caused by parasitic fungi, but only in the form of oral dosage. However, the low and variable bioavailability, gastrointestinal adverse effect and requirement of high concentration at the infection area call for alternative form, topical dosage form. But, due to its low solubility and skin permeability, it is difficult to make an effective topical dosage form containing itraconazole. If the eutectic mixture is used to prepare topical solution, the solubility and the flux of the insoluble drug will be increased. Therefore, as using eutectic mixture containing itraconazole and phenol, this problem was proved at this study.
The purpose of this study was to formulate the optimal topical solution containing itraconazole-phenol eutectic mixture and evaluate of permeation, deposition and irritation for topical skin delivery of itraconazole.
The effect of formulations on the hairless mouse skin permeation was determined using Franz diffusion cells at 37℃. The ratio of itraconazole-phenol eutectic mixture and the concentrations of itraconazole, penetration enhancer and thickening agents effected not only the skin permeation rate of itraconazole but also the lag time. As the results, increasing the concentration of itraconazole also increased the amount of itraconazole permeated and the flux of itraconazole through excised hairless mouse skin. But, depending on the results of stability test, the concentration of itraconazole was chosen as 5%. And by adding oleic acid in the topical solution, the flux of itraconazole was significantly increased. It was considered that oleic acid decreased the phase transition temperatures of the skin lipids with a resultant increase in motional freedom or fluidity of theses structures. Also, by increasing benzyl alcohol in the topical solution, lag time of itraconazole permeated was significantly increased. But faster lag time is more efficient. And by increasing concentration of HPMC 2910 in the topical solution, the viscosity of topical solution was increased and thus the flux of itraconazole was significantly decreased. It was considered that topical solutions of lower viscosity were more effective to permeate through skin layer to receptor phase. As a result, cumulative amount of itraconazole permeated through excised hairless mouse was 32.68 ± 4.39 µg/cm2 at optimal formulation.
And the effect of the optimal formulation and the former formulation without phenol on the Sprague-Dawley rats’ skin deposition was determined. At skin deposition test, there was no significant difference (P > 0.05) between optimal formulation and the same formulation but phenol at stratum corneum. It means that the deposition of itraconazole is not affected by eutectic mixture. And the reason of decreasing the amount of itraconazole deposited as time goes, was that the flux of itraconazole permeated through SD rats’ skin also was decreased. However, the results of dermis and epidermis, There was no significant difference at 4hr (P > 0.05), but there were significant differences at both 8 and 12 hr (P < 0.05). In particular, the formulation containing itraconazole-phenol eutectic mixture was presented the less amount of itraconazole deposited. It was considered that eutectic mixture given viscosity, such as reservoir, may control the release of itraconazole in topical solutions. And the reason of decreasing the amount of itraconazole deposited as time goes, was that the flux of itraconazole permeated through SD rats’ skin also was decreased. So, the rate of skin permeation may be less than the rate of metabolism.
The irritation test on New Zealand white rabbits was conducted. As the result, the topical solutions containing itraconazole-phenol eutectic mixture did not damage on the rabbits’ skin. Thus, this itraconazole topical solution containing itraconazole-phenol eutectic mixture may provide a useful topical delivery for itraconazole.

목차 (Table of Contents)

• I. INTRODUCTION 1
• I-1. Itraconazole 1
• I-1-1. Chemical structure of itraconazole 1
• I-1-2. Microbiology 4
• I-1-2-1. Mechanism of action 4
• I-1-2-2. Spectrum of activity 6
• I-1-3. Pharmacokinetics 6
• I-1-3-1. Absorption 6
• I-1-3-2. Distribution 7
• I-1-3-3. Metabolism 8
• I-1-3-4. Elimination 10
• I-1-4. Pharmacology 13
• I-1-4-1. Indications 13
• I-1-4-2. Adverse events 13
• I-1-4-3. Drug interactions 15
• I-1-4-4. Teratogenicity 17
• I-2. Topical delivery system 20
• I-2-1. Summary 20
• I-2-2. Structure of skin 21
• I-2-3. Skin permeability 25
• I-2-3-1. The barrier 25
• I-2-3-2. Routes of skin permeation 26
• I-2-4. Design of topical delivery system 27
• I-2-4-1. Solutions 28
• I-2-4-2. Powders 28
• I-2-4-3. Emulsions and microemulsions 29
• I-2-4-4. Ointments 29
• I-2-4-5. Gels 30
• I-2-4-6. Liposomes 31
• I-2-4-7. Aerosols 32
• I-3. Eutectic mixture 32
• I-3-1. Summary 33
• I-3-2. Application of eutectic mixtures in transdermal delivery 33
• I-4. Additives 37
• I-4-1. Phenol 37
• I-4-2. Skin permeation enhancer 39
• I-4-2-1. Benzyl alcohol 40
• I-4-2-2. Oleic acid 41
• I-4-3. Thickening agents 42
• I-4-3-1. HPMC 42
• II. OBJECTIVES 44
• III. EXPERIMENTAL METHODS 46
• III-1. Materials 46
• III-2. Quantitative analysis of itraconazole 47
• III-2-1. HPLC assay 47
• III-2-2. HPLC method 48
• III-2-3. HPLC system 48
• III-2-4. Preparation of stock solution 48
• III-2-5. Preparation of standard solution 49
• III-2-6. Validation of itraconazole HPLC analysis 49
• III-3. Preparation of itraconazole-phenol topical solutions 50
• III-4. Evaluation of itraconazole-phenol topical solutions 52
• III-4-1. Stability test 52
• III-4-2. In vitro skin permeation test 52
• III-4-3. Measurement of viscosity 53
• III-4-4. In vivo skin deposition test 55
• III-4-5. Skin irritation test 56
• III-5. Statistics 56
• IV. RESULTS AND DISCUSSIONS 59
• IV-1. Quantitative analysis of itraconazole 59
• IV-1-1. Validation of itraconazole method using HPLC 59
• IV-2. Stability test 63
• IV-3. In vitro skin permeation test 63
• IV-3-1. Effects of itraconazole concentration on the permeation of itraconazole through hairless mouse skin 63
• IV-3-2. Effects of oleic acid concentration on the permeation of itraconazole through hairless mouse skin 68
• IV-3-3. Effects of benzyl alcohol concentration on the permeation of itraconazole through hairless mouse skin 69
• IV-3-4. Effects of itraconazole-phenol ratio on the permeation of itraconazole through hairless mouse skin 76
• IV-3-5. Effects of HPMC 2910 concentration on the permeation of itraconazole through hairless mouse skin 79
• IV-4. Measurement of viscosity 83
• IV-5. In vivo skin deposition test 86
• IV-5-1. Deposition amounts of itraconazole on stratum corneum 86
• IV-5-2. Deposition amounts of itraconazole on dermis and epidermis 86
• IV-6. Skin irritation test 90
• IV-6-1. Irritation of the control 90
• IV-6-2. Irritation of the optimal formulation 90
• V. CONCLUSIONS 95
• Ⅵ. REFERENCES 98
• Ⅶ. 국문 요약 103