未來發展方向

基於目前所獲得的實驗結果，我們認為以藥物載體接合磁振造影顯影劑與一 些對分子標記有高親和力的物質當做分子探針，從事標靶影像是可行的。我們也 證實了使用標靶磁振造影顯影劑加上動態顯影磁振造影所提供的”三十分鐘顯 影劑流失比率”，具有早期偵測標靶治療藥物反應的潛力。整理未來研究發展方 向包括：

一、考慮進入人體實驗

本藥物載體之主幹為聚二乙醇 polyethylene glycol, 具有溶解度好安定，

生物相容性高，毒性低，在體內可避免被蛋白質吸收，避免被巨噬細胞吞噬，循 環時間長，細胞吸收效率高等優點，故已被廣泛應用於生物科技和醫學領域多年 (Harush-Frenkel, Bivas-Benita et al.)。其本身又為 biodegradable,不會堆 積於體內，故此聚二乙醇藥物載體應可考慮進入人體實驗階段，不然光是證明此 種標靶影像診斷方式於動物身上有用，對於目前癌症治療與病人福祉就毫無益 處。我們初步在動物身上所做的毒性分析，也證明其致死劑量ＬＤ50 甚高，是 ㄧ安全的藥物載體。未來將和專家研究進入人體試驗的可行性。

二、考慮將癌症治療藥物一同接合到藥物載體上

既然我們已證明此分子探針能在體內聚積於病灶附近，為何不趁此機會將治 療藥物一併帶過去？現在對於癌症治療藥物的設計有一新的共識就是朝向 multicomponent 發展，將化學治療藥物與多分子聚合體(polymer)接合，因藥物

-多分子聚合體分子量較大，可使藥物存留在體內的時間延長，逐漸釋放至體內，

使藥物注射的頻率可以減少，改善化學治療藥物 schedule dependent 的現象(藥 物毒性與藥物注射次數有正相關) (Scott, Yao et al., 2009)；multicomponent 藥物另ㄧ好處就是可接合和腫瘤表面接受體有高親和力的分子探針，以同時達到 標靶治療的目的。

我們初步已經將 Camptothecin 接合到此藥物載體。Camptothecin 是從中國 植物 Camptotheca acuminata 所提煉出的化學治療藥物，其作用機制在於抑制 DNA topoisomerase I，對於大腸癌，胃癌，非小型細胞肺癌，黑色細胞瘤皆有 明顯療效(Giovanella, Stehlin et al., 2002; Goldwasser, Shimizu et al., 1996; Natelson, Giovanella et al., 1996)，但因該藥物容易引起出血性膀胱 炎、胃炎，同時 CPT 亦有 schedule dependent 的效應，故使其臨床使用受到限 制。Conover 等人曾將 PEG 與 camptothecin 接合，運用在動物腫瘤模式上，獲 得明顯的療效，同時藥物副作用也減輕許多(Conover, Greenwald et al., 1998)，PEG-CPT 如今也已完成 phase II 臨床研究(Scott, Yao et al., 2009)，

使用於食道癌與胃癌病人，有部分病人腫瘤變小，藥物副作用也較減少。我們推 想既然 pegylated camptothecin 已經被證實有療效，那再加上一些對分子標記 有高親和力的物質當做分子探針在治療藥物的旁邊，使毒性高的化學治療藥物高 選擇性的聚集在腫瘤，是否能更加強藥物治療的療效，使身體正常組織受到較小 的細胞毒性影響，降低藥物副作用。如接合上葉酸以治療有大量表現葉酸接受體 的腫瘤，接合上皮細胞生長因子(epidermal growth factor, EGF)以治療臨床上 有大量表現上皮細胞生長因子接受體(EGFR)的腫瘤，或接合上環狀ＲＧＤ胜肽以 抑制腫瘤血管新生，都是未來值得繼續研究的目標。

三、繼續從事早期偵測其他標靶治療藥物療效的研究

本研究建立了一個有效評估標靶治療藥物療效的平台，今後我們可以將其他 在臨床上已經開始使用的標靶治療藥物應用在此平台，例如我們可以將上皮細胞 生長因子接受體(epidermal growth factorreceptor, EGFG)拮抗劑接合於ＰＥ

Ｇ藥物載體用以早期評估上皮細胞生長因子接受體標靶治療藥物 cetuximab 的 治療反應，我們也可以以同樣模式測試其他發展中具有潛力的標靶治療新藥。我 們也已經開始合成葉酸接受體標靶顯影劑，預備早期評估目前用於治療肺癌藥物 中抗葉酸藥物（anti-folate）alimta 之在大量表現葉酸接受體的 KB 細胞腫瘤 於動物腫瘤模式的治療療效。

遠 瞻 影 像 醫 學 的 未來，對於解剖構造變化的偵測仍將是會臨床診斷的基 本。但 發展分子影像學，嘗試瞭解患者體內功能性、生理性、分子產物的改變，

將其定性定量，將會開啟影像醫學的另一里程碑，使其可以協助臨床針對每一個 個體設計最適合的治療策略(individual therapy)，在影像上”看見”腫瘤是否 存在某藥物的特殊標的，進而決定該藥物是否適合用於該病人。目前許多癌症治 療藥物為 cytosatic 而非 cytofoxic，此時除非等到腫瘤大小發生改變，使用傳 統影像工具較不易判斷藥物治療療效，而分子影像有望在治療早期預測該藥物是 否有效。美國 NIH 將分子影像學列為五項通往發明的新途徑之一(new pathways to discovery, http:// nihroadmap.nih. gov)，其重要性可見一般。

論文英文簡述(summary)

Part 1. Blood Perfusion of Vertebral Lesions Evaluated With Gadolinium-Enhanced Dynamic MRI: In Comparison With Compression Fracture and Metastasis

Introduction

GADOLINIUM diethylenetriamine-pentaacetic acid (Gd- DTPA) shows pharmacokinetics similar to that of iodinized contrast media, and produces an increase of signal intensity (SI) in the T1-weighted spin-echo magnetic resonance (MR) image by reducing T1 relaxation time. It has been demonstrated that zones showing a marked increase in SI are correlated with highly vascularized regions, whereas zones with low or no increase in SI are often correlated with necrotic area in malignant tumors. In recent years, dynamic MRI has been used for evaluating musculoskeletal neoplasms and monitoring response to chemotherapy. Verstraete et al reported that first-pass data from dynamic contrast- enhanced MRI depicted tissue vascularization and perfusion of musculoskeletal neoplasms, although an overlap in the slope values was demonstrated in high vascular benign and malignant lesions. Van Der Woude et al also utilized dynamic MRI to detect residual viable tumor of postchemotherapy osteosarcomas and Ewing’s sarcoma before surgery. In 1997, Bollow et al used dynamic MRI to compare normal and malignant bone marrow infiltrations.

Thus, dynamic MRI proved to be an effective and noninvasive method for evaluating the in vivo blood perfusion of bone, marrow, and tumors.

MRI is highly useful for detecting diseases of the bone marrow, and has become the imaging modality of choice for marrow metastatic disease.

However, differential diagnosis of vertebral compression fracture

without obvious clinical or radiologic evidence of malignancy in an elderly patient presents a difficult problem. To our knowledge, there have been a limited number of articles evaluating bone marrow blood perfusion in normal or diseased vertebrae. Bone marrow perfusion evaluated with gadolinium-enhanced dynamic MRI was reported by Cova et al and showed a strong correlation between MR data and microsphere blood-flow

measurements. Therefore, our study investigated the peak contrast enhancement percentage, enhancement slope, and time-intensity curve (TIC) patterns in the first pass of contrast into diseased vertebral bodies to analyze the perfusion characters of compression fracture and metastatic vertebrae with and without pathologic fractures.

Results

The acute compression fracture in group I subjects had a higher peak enhancement percentage compared to chronic compression fracture in group II subjects (213.82 _193.07 vs. 55.24 =47.98; P =0.05). Metastatic vertebrae in group III subjects had a higher peak enhancement percentage as compared to chronic compression fracture in group II (152.52 =89.42 vs. 55.24 =47.98; P =0.001). Pathologic fracture subjects in group IV had a higher peak enhancement percentage as compared to chronic compression fracture in group II (136.74 _51.37 vs. 55.24 =47.98; P =0.05). Acute compression fracture in group I subjects revealed a higher enhancement slope compared to chronic compression fracture in group II subjects (8.27

=9.9 vs. 1.94 =2.31; P =0.05). Metastatic vertebrae in group III subjects had a higher enhancement slope as compared to chronic compression fracture in group II (7.7 =5.11 vs. 1.94 =2.31; P =0.05). Pathologic fracture subjects in group IV had higher enhancement slope as compared to chronic

compression fracture in group II (8.13 =4.54 vs. 1.94 =2.31; P =0.05).

No significant difference for peak enhancement percentage and enhancement slope was noted in comparison with acute compression fracture and metastatic vertebrae with or without fracture, nonfractured metastatic vertebrae, and pathologic vertebral fracture.

Patterns of TIC

The data were reviewed by two radiologists on separate occasions, and consensus was obtained for the curve pattern classification. All 12 type D curves (100%) were found in malignant groups (groups III and IV). Six (85.7%) of seven type E curves were revealed in benign compression fracture groups (groups I and II). Type C was the most common type in malignant groups (19/38, 50%), and the second-most common type in benign compression fracture (10/33, 30.3%). The type A curve was found in chronic compression fracture cases (4/21, 19%). The type B curve was found in benign compression fracture (13/33, 36.3%) and nonfractured metastatic vertebrae (5/32, 15.6%).

DISCUSSION

Several factors contribute to the contrast enhancement of tissue: 1) a vascular supply, 2) an interstitial space for sequestering the contrast material, and 3) a route for the contrast material getting out of the vasculature. The rapid rise in SI immediately after the injection reflects the presence of gadopentetate dimeglumine in intravascular space as well as an increasing contribution of gadopentetate dimeglumine in

extracellular space. An equilibration phase may be reached if there is a balance between the intra- and extravascular compartments, which represents the plateau of the type C curve. The type C curve was seen in

most of the normal vertebrae, benign compression fractures, and malignant vertebral lesions (Table 3). Therefore, a type C curve is not helpful for differentiating between benign and malignant vertebral lesions.

In a pilot study, we performed a contrast-enhanced dynamic pulse sequence in some cases for a longer observation time (280 seconds). The major event in the late phase was a gradual wash-out of contrast from tissue, which was observed in both compression-fracture and malignant groups, so we decided to focus on the “first-pass” and early-phase contrast

enhancement and wash-out patterns.

There were in total 13 type D curves (rapid contrast wash-in followed by early washout), presented in 71 measurements All of the type D curves were found in metastatic disease with or without associated pathologic fracture. We considered that the early washout phenomenon is specific for differentiating metastatic disease from benign compression fracture, because no type D curve occurred in the latter group. However, since the type D curve was found in only one-third (13/38, 34.2%) of malignant vertebral lesions, the sensitivity of the type D curve for diagnosing malignant vertebral lesions is not sufficiently high. The early wash-out pattern was previously described by Van Der Woude et al. This pattern was encountered in malignant bone tumors with high cellularity. The areas were histopathologically characterized by packed viable tumor cells with little intercellular matrix. A rapid contrast wash-in is followed by early wash-out because of the small volume of the interstitial space in areas consisting of packed viable tumor cells with a scarcity of matrix.

Subsequently, SI decreases because of a lower concentration of

gadopentetate demeglumine inflow. Early wash-out of contrast was also

revealed in breast cancer studies by Daniel et aland Kuhl et al using Gd-DTPA-enhanced dynamic MRI, in which it was concluded that the washout phenomenon is specific for discriminating between benign and malignant disease.

There were seven type E curves, presented in 71 measurements. Most of the type E curves (6/7, 85.7%) were found in benign compression fracture cases (groups I and II). The fracture healing process has been arbitrarily divided into four phases: callus proliferation, vascularization, calcification, and reorganization. These phases overlap and can also be classified as an inflammatory phase, reparative phase, and a remodeling phase. An increase in vascularity at the fractured site is seen in the inflammatory phase, associated with infiltration of inflammatory cells, vasodilatation, and exudation of plasma. Due to an increase in vessel permeability in the inflammatory reaction, the type E TIC might be explained by more contrast sequesters into extracapillary space in the inflammatory phase without a balanced washout through venous drainage.

A type E TIC was also revealed previously by Konig et al. By using a fast gradient-echo Gd-DTPA-enhanced dynamic MR pulse sequence, a second rising slope was shown in the hypervascular pannus of rheumatoid arthritis. The pannus is a highly inflammatory synovial tissue characterized by edema, infiltration of inflammatory cells, hypertrophy of synovial lining cells, and proliferative granulation tissues. We hypothesized that the cause of a second rising slope might be due to increased contrast sequestering into extracapillary space in an inflammatory event, and that type E is preferential for benign disease processes. However, since only six out of 33 (18.2%) vertebral compression fractures showed a type E curve, the

sensitivity of the type E curve for diagnosing benign vertebral

compression fractures is low. There was no significant difference for peak enhancement percentage and enhancement slope between benign acute compression fracture, metastatic vertebral lesion, and pathologic compression fracture. There were overlapping areas between these groups, which will cause diagnostic difficulty only when their peak enhancement percentage and slope are examined. According to Erlemann’s and

Verstraete’s study, an overlap in the slope values was also demonstrated in the high vascular benign and malignant lesions.

In the early inflammatory phase of fracture healing, an increase in vascularity 20 times greater than the resting circulation at the fractured site has been reported. Thus, high peak enhancement percentage and enhancement slope were observed in benign acute compression fracture.

When conventional MR images for acute benign compression fracture cases were reviewed, the recently collapsed vertebral bodies all showed marked bone marrow edema in T2-weighted images, and the evidence of hyperemia was also observed from post-enhanced MRI. The proliferation of

vascularity during fracture healing lasts about 3–10 weeks. In our study, the peak enhancement percentage and enhancement slope in acute

compression fracture were significantly higher than that of the chronic compression fracture group, corresponding well to the fracture healing process. Several manifestations of MRI have been proposed as useful adjuncts in differentiating benign from malignant vertebral fracture. The findings that were preferential for a pathologic fracture were: complete marrow replacement, paravertebral soft-tissue lesion, pedicle

involvement, abnormal expansile lesion with illdefined margin, and strong

heterogeneous or irregular nodular-type soft-tissue infiltration.

However, diagnostic difficulties may be encountered in some cases. The Gd-DTPA-enhanced dynamic MR study may provide additional information in equivocal cases.

In conclusion, low enhancement percentage and shallow slope are more suggestive of a benign vertebral lesion. A type E curve (rapid contrast wash-in with a second rising slope) is more likely to occur in benign compression fracture. A type D curve (rapid contrast wash-in with early wash-out phenomenon) is preferential for metastatic disease of vertebra with or without associated pathologic fracture. Dynamic Gd-DTPA-enhanced MRI provides useful information for differentiating between benign and malignant vertebral lesions in certain cases.

‘

Part 2. Dynamic Contrast-Enhanced Folate-Receptor-Targeted MR Imaging

Part 2. Dynamic Contrast-Enhanced Folate-Receptor-Targeted MR Imaging