Clinical Pharmacy & Pharmacogenomics Life

The Pharmacogenetics of Metformin & Its Impact on Plasma Metformin Steady-state Levels & Glycosylated Hemoglobin A1c.

Pharmacogenet Genom. 2011;21: 837-850

Link: http://www.ncbi.nlm.nih.gov/pubmed/21989078

首先
我們要來回顧一下這個從 1975 年上市至今的老藥 – Metformin (Glucophage)

在 T2DM 的治療中，Metformin 扮演了很重要的腳色，近十年的研究報告指出，其可以降低肥胖者發生糖尿病的罹病率與死亡率。Metformin 雖能降低血糖值，但發生低血糖的風險卻很低，此外，metformin 也能使體重降低。Metformin 較常見的副作用包括了腸胃道的不適感，以及較少見的乳酸中毒 (lactic acidosis)。
Metformin has been known for nearly a century and has experienced a renaissance in the treatment of type 2 diabetes during the recent decade because of the documentation that the drug reduces morbidity and mortality in obese type 2 diabetics. In addition to its blood glucose-lowering effect, metformin induces a small weight reduction and has only a minimum risk of inducing hypoglycemia. The drug frequently induces gastrointestinal side effects but very rarely causes lactic acidosis.

Metformin 實際之分子生理機轉至今仍尚未完全明瞭，但目前被認為可能與 serine-threonine kinase 11 pathway 及活化 AMP-activated protein kinase 有關。Metformin 可提升周邊組織對 insulin 的敏感性，提升葡萄糖的使用率，並能降低肝臟糖質新生作用 (gluconeogenesis)。
The exact molecular mechanism of antidiabetic effects of metformin has not yet been fully elucidated but it seems to involve the serine-threonine kinase 11 pathway and activation of the adenosine monophosphate-activated protein kinase. Metformin increases the peripheral insulin sensitivity, increases the peripheral uptake of glucose, and decreases the gluconeogenesis in the liver.

Metformin 本身是個強鹼，而在生理環境下的幾乎皆以陽離子態存在 (>99.9%)。因此其需要轉運蛋白 (transporter) 的運送以穿越細胞膜。Metformin 在腸道呈現劑量依賴型吸收模式。
Metformin is a strong base and at physiological pH, it exists virtually only (> 99.9%) in its cationic form. Thus, its passage across cell membranes is heavily dependent on transporters. The intestinal absorption of metformin is dose dependent.

再來是登場的是...
那些年...  跟Metformin 一起作用的基因...

漿膜單胺轉運蛋白 (plasma membrane monoamine transporter, PMAT) 是屬於 solute carrier (SLC) 第 29 家族的一員。其主要分布於小腸及腎小管的頂端膜，並可能負責調控腸道對 metformin 的吸收作用。
The plasma membrane monoamine transporter (PMAT) is a recently discovered proton-activated transporter belonging to the solute carrier (SLC) family 29. PMAT is located in the apical membrane of the epithelial lining of the small intestine and renal tubules, and it probably mediates the intestinal uptake of metformin.

OCT1 (SLC22A1) 與 OCT2 (SLC22A2) 是多功能的 OCTs 轉運蛋白，此二者分別廣泛分布於肝臟竇狀細胞以與腎小管細胞的基底膜。其中 OCT1 亦坐落於肝細胞及腎小管細胞的基底膜。
此二者皆為單一輸送方向的轉運蛋白，並負責調控 metformin 運送至標的細胞之促進型擴散作用，並能促進 metformin 自血液中被輸送至肝臟。
OCT1 (SLC22A1) and OCT2 (SLC22A2) are poly-functional OCTs located predominantly at the sinusoidal cells of the liver and basolaterally in the kidney tubular cells, respectively. OCT1 is also expressed basolaterally in the enterocytes and in the renal tubular cells. They are both uniporters that mediate facilitated diffusion of metformin in either direction in the target cells and hence are the major determinants of the hepatic uptake of metformin (OCT1) and the renal uptake (OCT2) from the blood.

MATE1 與 MATE2 又名 SLC47A1 與 SLC47A2，此二者是腎小管細胞頂端膜之氫離子/藥物交換轉運蛋白，並負責將 metformin 擴散排除至尿液中。
The multidrug and toxin extrusion transporters 1 (SLC47A1) and 2 (SLC47A2), alias MATE1 and MATE2, are located in the apical side of the renal tubular cells; they are H+/drug antiporters that facilitate extrusion of metformin through the urine.

試驗目的：

這五個基因與 metformin 之生體清除率息息相關 (~90%)，故本篇作者希望能藉由此大型試驗，探討這些基因上的多型性與 metformin 穩定狀態血中濃度和 HbA1c 控制的相關性。

實驗設計：

這是來自於 SDDS 的實驗 ［2 x 2 x 2 factorial, prospective, randomized, partly blinded, placebo-controlled, multicenter study］，所收納的 371 位病人都是丹麥人。
The South Danish Diabetes Study (SDDS) has recently been described in detail in a separate article. In brief, SDDS was designed as a 2 x 2 x 2 factorial, prospective, randomized, partly blinded, placebo-controlled, multicenter study comprising 371 Danish individuals with type 2 diabetes in eight parallel groups.

受試者會在一年內安排 15 次的回診，並在第 8, 9 , 10 次回診 (第 3, 6, 9 個月) 測量 metformin 穩定狀態血中波谷濃度 (trough)，並在第 1, 9, 15 次回診 (第 0, 6, 24 個月) 測量其糖化血色素 (HbA1c)。
The patients were treated per protocol for 24 months including 15 visits. For this study, the steady-state measurements of trough plasma metformin levels were obtained at visits 8, 9, and 10 (at 3, 6, and 9 months) and for HbA1c at visits 1, 9, and 15 (at 0, 6, and 24 months).

實驗對象：

受試者之納入條件為：年齡介於 30 – 70 y/o，空腹 C-peptide > 300 pmol/L，BMI > 25 kg/m2，患有糖尿病超過兩年，HbA1c 介於 8.0% - 12.0% 之間。
The inclusion criteria were: age of 30–70 years, fasting C-peptide of more than 300 pmol/l, BMI of more than 25 kg/m2, diabetes for more than 2 years, and 8.0% < HbA1c < 12.0%.

受試者之排除條件為：Scr > 120 mcmol/L，ALT/AST 超過正常值上限的 2.5 倍，總膽固醇 (TC) > 10 mmol/L，總三酸甘油脂 (TG) > 8 mmol/L，血紅素 (Hb) 低於正常值，之前曾使用 glitazone 類藥物超過 30 天，NYHA III or IV，懷孕者，視力惡化者，對低血糖徵狀沒有概念者，精神耗弱，酒精成癮，具有器官或系統性重大疾病者，無法控制之高血壓者 (> 180/110 mmHg)，併用類固醇，嚴重肺疾病，惡性腫瘤病史者。
Whereas the exclusion criteria included: intolerance to metformin/glitazones, s-creatinine of less than 120 mmol/l, serum alanine aminotransferase/ serum aspartate aminotransferase, 2.5 X upper  normal  limit, total cholesterol of more than 10 mmol/l, total triglyceride of more than 8 mmol/l, hemoglobin of less than normal range, treatment with glitazone preceding 30 days, NYHA III or IV, night work, pregnancy, poor vision, unawareness of hypoglycemia, mental sickness, alcohol abuse, clinically relevant major organ or systemic illness, uncontrolled hypertension of more than 180/110 mmHg, systolic or diastolic, steroid treatment, severe lung disease, and history of malign disease.

SNPs 選擇條件：

1. 為這五個轉運蛋白基因上的轉錄區域 (coding region)
2. 之前曾有研究指出可能會影響 metformin 臨床藥效的位點 (study-based)
3. 在高加索人種中具有基因多型性現象之位點 (polymorphism in Caucasian)
4. PMAT 此基因使用了目前已知的誤義變異 (nonsynonymous) 並進行 tagging SNPs。

Selection of the relevant genetic variants was based on the following criteria: (a) the genetic variation had to be located in a gene coding for a transporter of metformin, (b) genetic variation influencing the transport of metformin or the ability to reach a relevant clinical endpoint ex HbA1c during metformin therapy had to be reported in the literature, (c) the genetic variation had to be present in Caucasians, and (d) PMAT was examined as hypothesis generating including known nonsynonymous SNPs and tagging SNPs (tagSNPs).

實驗結果：　　　　　（僅摘錄重點）

Baseline:

 1. 共有 159 個病人完成實驗，所有病人皆服用 1 g metformin, twice daily。
2. 所有病人的平均採血時間為。
3. 病患的穩定狀態波谷血中濃度為 576 ng/L (95% CI: 520-637)
　全距為 (54-4133 ng/L) --> 差異近 80 倍之譜，顯示個體差異變化非常大。
4. HbA1c 的變化為：
　　六個月內的變化為：-1.5% to -2.0%
　　一年內的變化為：-1.3% to -2.0%

PGx:

基因型與濃度之分析：

1. 在五個基因中，僅有 OCT1 rs72552763 此位點與濃度具有顯著相關性，在此位點帶有變異的缺失對偶基因 (del) 越多，其血中濃度越低 (p=0.02 in the trend test)：

　　Wt/Wt: 624 ng/mL (95%CI: 550-710)
　　Wt/Var: 499 ng/mL (95%I: 418-596)
　　Var/Var: 346 ng/mL (95%CI: 76-1567)

在使用顯性模式的分析中，也發現了帶有野生型同質基因型患者 (wild-type homozygous) 與異質性基因型患者 (heterozygous) 間，在血中濃度的比較上具有顯著差異 (p=0.027)。

2. 進一步將 OCT1 上的四個位點依照之前的研究，以功能性基因型組合模式作分析，發現若是病患本身為帶有較多降低功能之基因型的 haplotype 者，則血中濃度會顯著的降低 (p=0.001)。

基因型與 HbA1c 之分析：

1. 在五個基因中，僅發現 OCT1 rs34130495 在六個月的 HbA1c 變化中，帶有 heterozygous 的患者其降低 HbA1c 的能力顯著較差 (p=0.003)；但在一年的 HbA1c 變化中，此趨勢不再顯著 (p=0.16)。

2. 在功能性基因型組合模式分析中，野生型同質基因型者其 HbA1c 降低的程度會顯著的優於變異型同質基因型者 (p=0.004)。

討論與結論：

1. OCT1 若發生變異則可能導致其本身轉運功能下降，除了可能使得 metformin 較不易進入肝臟細胞並發揮作用外，亦可能會減少腸胃道對 metformin 的吸收，並增加腎臟對 metformin 的清除能力，致使血中波谷濃度下降，進而致使療效降低。

2. 然而其他的如調控 metformin 分布之 SLC 轉運蛋白，其本身可能存在著較複雜的交互作用，因此需要近一步的實驗以探討其相關性。

3. OCT1 若發生變異可能導致活性改變，其不只會影響到 metformin 的穩定狀態波谷血中濃度，也會對其長期的 HbA1c 控制目標產生影響。

Wind Wei-Lun Chang

2011.11.16