<title lang="zh">用於抗發炎之套組</title><title lang="en">KIT FOR INHIBITING INFLAMMATION</title><technical-field><p>本發明係關於一種用於抗發炎之套組，其係包含（1）一第一部分，其係一容器，具一容置空間且可避光；以及（2）一第二部分，其係一均勻溶液，包含（a）玻尿酸及（b）一3-羥基-3-甲基戊二醯輔酶A（HMG-CoA）還原酶抑制劑，其中，該第二部分係存在於該容置空間內且與該第一部分接觸。較佳地，該第二部分係一抗發炎醫藥組合物，其中，以成分（a）與成分（b）之總重計，成分（a）之含量為約98.5重量%至99.5重量%，成分（b）之含量為約0.5重量%至1.5重量%。</p></technical-field><background-art><p>關節炎係一常見之慢性疾病，其係因關節軟骨退化或結締組織發炎而造成關節疼痛，進而影響關節的正常活動。依據發生部位與原因，關節炎共可分為一百多種類型，最常見者包括骨關節炎（退化性關節炎）、類風濕性關節炎、痛風性關節炎、細菌性關節炎、僵直性脊椎關節炎、及紅斑性狼瘡等。</p><p>針對關節炎之治療，一般係於初期先採取保守性、非手術性的療法，例如藥物治療與注射劑治療，當初期治療未見成效時，才會採用外科手術的治療方式。已知藥物治療包括使用類固醇與非類固醇之抗發炎藥物，其中，類固醇藥物雖具止痛效果快且顯著之優點，但會引起許多副作用，例如骨質疏鬆症、傷口不易癒合、上消化道出血，甚至會加重如高血壓或糖尿病等疾病之症狀。至於非類固醇藥物，雖亦具良好的止痛效果，但若長期使用，則會產生諸如消化性潰瘍、下肢水腫及腎臟功能損傷等副作用。</p><p>玻尿酸又稱「透明質酸」或「醣醛酸」，已知其具抗發炎效果，故廣泛運用於抑制骨關節炎之注射劑中，例如，將含有玻尿酸之注射液直接注射至患者之關節，以適度減緩發炎及病患之疼痛感。此外，玻尿酸亦可作為幫助關節活動的潤滑劑，同時達到改善關節功能的效果。然而，業經發現，玻尿酸雖可有效減輕疼痛，但其進入人體後，有時反而會在數日內引起暫時性發炎反應，甚至造成慢性發炎，不利於所欲提供之抗發炎效益（此可參見Leopold et al., Increased frequency of acute local reaction to intra-articular hylan GF-20 (Synvisc) in patients receiving more than one course of treatment.
<i>J Bone Joint Surg</i>, 2002;84: 1619-23；Bernardeau et al., Acute arthritis after intra-articular hyaluronate injection: onset of effusions without crystal.
<i>Ann Rheum Dis</i>, 2001;60:518-20；以及Kroesen et al., Induction of an acute attack of calcium pyrophosphate dihydrate arthritis by intra-articular injection of hylan G-F 20 (Synvisc).
<i>Clin Rheumatol</i>,2000;19:147-9，該等文獻之全文併於此處以供參考）。因此，若能以較低玻尿酸劑量提供所欲之抗發炎效果，則可減緩、甚至避免玻尿酸進入人體之後所可能引起之後續發炎反應。
</p><p>本案發明人發現，透過結合使用玻尿酸與相對極少量之HMG-CoA還原酶抑制劑，可達到一不可預期之增進的抗發炎效果，有效降低玻尿酸之用量，從而減輕或免除玻尿酸所引起之暫時性發炎。</p><p>雖然高分子量玻尿酸相較於低分子量玻尿酸具有較佳之抗發炎效果，但本案發明人研究發現，若將3-羥基-3-甲基戊二醯輔酶A（下文簡稱為「HMG-CoA」）還原酶抑制劑與高分子量玻尿酸混合提供一均勻溶液，則其中之HMG-CoA還原酶抑制劑容易自該溶液中析出，無法穩定存在。經進一步研究發現，前述由HMG-CoA還原酶抑制劑與高分子量玻尿酸混合提供之均勻溶液，可以穩定存在於避光環境下，有利於該溶液在臨床上的應用。</p></background-art><disclosure><p>本發明之一目的，在於提供一種用於抗發炎之套組，其係包含（1）一第一部分，其係一容器，具一容置空間且可避光；以及（2）一第二部分，其係一均勻溶液，包含（a）玻尿酸及（b）一3-羥基-3-甲基戊二醯輔酶A（HMG-CoA）還原酶抑制劑，其中，該第二部分係存在於該容置空間內且與該第一部分接觸。該第二部分中之成分（a）的平均分子量較佳為約30萬至600萬道爾吞（Dalton），更佳為約200萬至600萬道爾吞。較佳地，該第二部分中之成分（b）係選自以下群組之至少一者：阿托法史坦酊（Atorvastatin）、薛利伐史坦酊（Cerivastatin）、氟伐史坦酊（Fluvastatin）、樂瓦史坦酊（Lovastatin）、美伐史坦酊（Mevastatin）、匹他伐史坦酊（Pitavastatin）、普伐史坦酊（Pravastatin）、辛維史坦酊（Simvastatin）、及瑞舒伐史坦酊（Rosuvastatin）。</p><p>本發明之另一目的，在於提供一種醫藥組合物，其係包含（a）玻尿酸、及（b）一3-羥基-3-甲基戊二醯輔酶A（HMG-CoA）還原酶抑制劑，其中，以成分（a）與成分（b）之總重計，成分（a）之含量為約98.5重量%至99.5重量%，成分（b）之含量為約0.5重量%至1.5重量%。其中，成分（a）的平均分子量較佳為約30萬至600萬道爾吞（Dalton），更佳為約200萬至600萬道爾吞；成分（b）較佳係選自以下群組之至少一者：阿托法史坦酊（Atorvastatin）、薛利伐史坦酊（Cerivastatin）、氟伐史坦酊（Fluvastatin）、樂瓦史坦酊（Lovastatin）、美伐史坦酊（Mevastatin）、匹他伐史坦酊（Pitavastatin）、普伐史坦酊（Pravastatin）、辛維史坦酊（Simvastatin）、及瑞舒伐史坦酊（Rosuvastatin）。前述醫藥組合物可作為本發明套組之第二部分，且視需要可進一步包含（c）一醫藥可接受載劑。</p><p>本發明之詳細技術內容及部分具體實施態樣，將描述於以下內容中，以供本發明所屬領域具通常知識者據以明瞭本發明之特徵。</p></disclosure><mode-for-invention><p>本發明之詳細技術及較佳實施態樣，將描述於以下內容中，以供本發明所屬領域具通常知識者據以明瞭本發明之特徵；惟，在不背離本發明精神下，本發明尚可以多種不同形式之態樣來實踐，不應將本發明保護範圍解釋為限於說明書所陳述者。此外，除非文中有另外說明，於本說明書中（尤其是在後述專利申請範圍中）所使用之「一」、「該」及類似用語應理解為包含單數及複數形式。</p><p>玻尿酸係一不具硫元素之糖胺聚醣，其基本結構是由兩個雙糖單位D-葡萄糖醛酸及N-乙醯葡糖胺組成的大型多醣類，具如下式（I）之化學式：
<img he="110" wi="222" img-format="jpg" id="i0002" img-content="drawing" orientation="portrait" inline="no" file="TWI663979B_D0001.tif" />（I）。
</p><p>玻尿酸係構成細胞外基質之主要成分之一，廣佈於內皮組織、結締組織、表皮組織及神經組織中，對於細胞之增生及遷移等生理活動至關重要，且為皮膚真皮層內之重要保濕成分，具有優異之黏容性與伸縮性，是一種非常理想的填充物質，故亦常用於美容產品及整形手術中。目前市售之玻尿酸溶液（例如含玻尿酸鈉水溶液之製劑）皆係以透明容器盛裝，常溫下之架儲期為三年。</p><p>本案發明人發現，於避光環境下儲存含有溶解有HMG-CoA還原酶抑制劑之高分子量玻尿酸溶液，可避免HMG-CoA還原酶抑制劑自高分子量玻尿酸中析出，有效提高該均勻溶液於架儲期內之穩定性，有利於臨床上之應用。因此，本發明係提供一種用於抗發炎之套組，其係包含（1）一第一部分，其係一容器，具一容置空間且可避光；以及（2）一第二部分，其係一均勻溶液，包含（a）玻尿酸及（b）一HMG-CoA還原酶抑制劑，其中，該第二部分係存在於該容置空間內且與該第一部分接觸。</p><p>於根據本發明之套組中，作為該第一部分之容器係瓶、罐、或袋，其可以任何合宜之材質提供，並無特殊限制。</p><p>關於作為該第一部分的可避光容器，所謂「可避光」係指可以遮蔽波長為290至450奈米（nm）之光線，使該波長範圍內之任一波長的穿透度不超過10%。此即，該可避光容器對於波長在290至450奈米（nm）之任一波長的光線的遮蔽程度達至少90%。適合用以提供該可避光容器之材料，可以參見例如美國藥典（United States Pharmacopeial Convention,
<i>United States Pharmacopeia 29</i>, United States Pharmacopeial Convention, Rockville, Md, USA, 2012），前述藥典之全文係併於此處以供參考。舉例言之，但不以此為限，可以使用I型玻璃（Type I glass）、II型玻璃（Type II glass）、III型玻璃（Type III glass）、NP型玻璃（Type NP glass）、或第一類至第六類塑膠（Plastic classes I-VI）以提供該作為第一部份之可避光容器。
</p><p>於根據本發明之套組中，作為第二部分之成份（a）的玻尿酸較佳係具平均分子量約30萬至600萬道爾頓（Dalton），更佳具平均分子量約200萬至600萬道爾頓。</p><p>適用於根據本發明之套組的玻尿酸，係可由天然物質製備而得（例如，萃取自雞冠、臍帶、軟骨及皮膚等活體部分），亦可由化學合成或利用酵母菌等微生物藉由培養或基因工程方法生產。亦可使用市售之玻尿酸製劑以提供根據本發明之套組，例如含玻尿酸鈉水溶液之製劑，包括ARTZ Dispo
<sup>®</sup>、SYNVISC
<sup>®</sup>、HYALGAN
<sup>®</sup>、及ORTHOVISC
<sup>®</sup>等，或包含交聯玻尿酸衍生物水溶液之製劑，包括SYNVISC
<sup>®</sup>及DUROLANE
<sup>®</sup>。
</p><p>適用於根據本發明之套組的玻尿酸，可為不形成鹽之游離狀態，亦可形成藥學上可容許之鹽。玻尿酸於藥學上可容許之鹽包括與無機鹼所形成之鹽，例如鈉鹽或鉀鹽等鹼金屬離子鹽、鎂鹽或鈣鹽等鹼土金屬離子鹽、以及銨鹽等；或者與有機鹼所形成之鹽，例如與二乙醇胺、環己胺或胺基酸等所形成之鹽。</p><p>於根據本發明之套組中，作為第二部分之成份（b）係一HMG-CoA還原酶抑制劑，係包括一類稱為「史坦酊（statins）」之藥物，該「史坦酊（statins）」藥物包含如下表所示之化合物：
<tables><table border="1" bordercolor="#000000" width="85%"><tbody><tr><td> 名稱 </td><td> 化學式 </td><td> 產品名 </td></tr><tr><td> 阿托法史坦酊 （Atorvastatin） </td><td><img wi="216" he="110" file="02_image004.tif" img-format="tif"/></img></td><td> Lipitor或Torvast </td></tr><tr><td> 薛利伐史坦酊 （Cerivastatin） </td><td><img wi="199" he="117" file="02_image006.tif" img-format="tif"/></img></td><td> Lipobay或Baycol </td></tr><tr><td> 氟伐史坦酊 （Fluvastatin） </td><td><img wi="155" he="163" file="02_image008.tif" img-format="tif"/></img></td><td> Lescol或Lescol XL </td></tr><tr><td> 樂瓦史坦酊 （Lovastatin） </td><td><img wi="170" he="151" file="02_image010.tif" img-format="tif"/></img></td><td> Mevacor、Altocor或Altoprev </td></tr><tr><td> 美伐史坦酊 （Mevastatin） </td><td><img wi="149" he="151" file="02_image012.tif" img-format="tif"/></img></td><td> 無 </td></tr><tr><td> 匹他伐史坦酊 （Pitavastatin） </td><td><img wi="193" he="121" file="02_image014.tif" img-format="tif"/></img></td><td> Livalo或Pitava </td></tr><tr><td> 普伐史坦酊 （Pravastatin） </td><td><img wi="185" he="155" file="02_image016.tif" img-format="tif"/></img></td><td> Pravachol、Selektine或Lipostat </td></tr><tr><td> 辛維史坦酊 （Simvastatin） </td><td><img wi="144" he="136" file="02_image018.tif" img-format="tif"/></img></td><td> Zocor或Lipex </td></tr><tr><td> 瑞舒伐史坦酊 （Rosuvastatin） </td><td><img wi="222" he="121" file="02_image020.tif" img-format="tif"/></img></td><td> Crestor </td></tr></tbody></table></tables></p><p>因此，於根據本發明之套組的部分較佳實施態樣中，作為第二部分之成分（b）係選自以下群組之至少一者：阿托法史坦酊（Atorvastatin）、薛利伐史坦酊（Cerivastatin）、氟伐史坦酊（Fluvastatin）、樂瓦史坦酊（Lovastatin）、美伐史坦酊（Mevastatin）、匹他伐史坦酊（Pitavastatin）、普伐史坦酊（Pravastatin）、辛維史坦酊（Simvastatin）、及瑞舒伐史坦酊（Rosuvastatin）。</p><p>本發明另提供一種抗發炎醫藥組合物，其係包含（a）玻尿酸及（b）一HMG-CoA還原酶抑制劑，其中該玻尿酸與HMG-CoA還原酶抑制劑均如上述說明。該醫藥組合物可做為根據本發明所提供之套組的第二部分，且可以呈任何合宜之形式，並無特殊限制。舉例言之，但不以此為限，該醫藥組合物可以皮下注射、靜脈注射、或關節內注射之形式提供。其中，視使用形式及用途而定，該醫藥組合物可進一步包含一醫藥可接受載劑，其中，該載劑包括賦形劑、稀釋劑、輔助劑、安定劑、吸收延遲劑、崩散劑、增溶劑、乳化劑、抗氧化劑、黏合劑、結合劑、增黏劑、分散劑、懸浮化劑、潤滑劑、吸濕劑等。</p><p>以適於皮下注射、靜脈注射、或關節內注射之針劑或點滴劑為例，可於根據本發明所提供之醫藥組合物中含有一或多種例如等張溶液、鹽類緩衝液（如磷酸鹽緩衝液或檸檬酸鹽緩衝液）、增溶劑、乳化劑、5%糖溶液、以及其他載劑等成分，以靜脈輸注液、乳劑靜脈輸注液、乾粉注射劑、懸液注射劑、或乾粉懸液注射劑等劑型提供該醫藥組合物。或者，可將該醫藥組合物製備成一注射前固體，以可溶於其他溶液或懸浮液中之劑型、或可乳化之劑型提供該注射前固體，並於投予至有需要之個體之前，將該注射前固體溶於其他溶液或懸浮液中或將其乳化，提供所欲之注射劑。</p><p>視需要地，可於根據本發明所提供之抗發炎醫藥組合物中另含有合宜用量之添加劑，例如緩衝劑、保存劑、防腐劑、抗菌劑、抗真菌劑等。此外，於該醫藥組合物中可視需要更包含一或多種其他活性成分，以進一步加強該醫藥組合物之功效或增加其運用靈活性與調配度，只要該其他活性成分對本發明醫藥組合物之效益沒有不利的影響即可。該其他活性成分之例子包括，但不限於，類固醇抗發炎藥物、非類固醇抗發炎藥物、葡萄糖胺（glucosamine）等。</p><p>於根據本發明所提供之抗發炎醫藥組合物中，成分（a）與成分（b）之配比並無特殊限制。一般而言，以成分（a）與成分（b）之總重計，成分（a）之含量為約80重量%至99.9重量%，且成分（b）之含量為約0.1重量%至20重量%。較佳地，以成分（a）與成分（b）之總重計，成分（a）之含量為約98重量%至99.9重量%，且成分（b）之含量為約0.1重量%至2重量%。更佳地，以成分（a）與成分（b）之總重計，成分（a）之含量為約98.5重量%至99.5重量%，且成分（b）之含量為約0.5重量%至1.5重量%。</p><p>由於本發明醫藥組合物可提供增進之抗發炎效果，故可用於抑制發炎，尤其可用於抑制關節炎，包括骨關節炎（退化性關節炎）、類風濕性關節炎、痛風性關節炎、細菌性關節炎、僵直性脊椎關節炎、及紅斑性狼瘡等，較佳係用於抑制骨關節炎及類風濕性關節炎。於本發明之一實施態樣中，係將本發明醫藥組合物製成注射劑之型式，以用於關節注射，治療關節炎。</p><p>端視投予個體之需求、年齡、體重、及健康況狀，係可以一日一次、一日多次、或數日一次等不同投藥頻率將根據本發明所提供之抗發炎醫藥組合物施用至有需要的個體。於根據本發明所提供之醫藥組合物中，可視實際應用需求，調整成分（a）與成分（b）之含量比例。</p><p>如後附實施例所示，本案發明人發現，透過結合使用玻尿酸與相對極少量之HMG-CoA還原酶抑制劑（例如，以玻尿酸與HMG-CoA還原酶抑制劑之總重計，含量為約0.5重量%至1.5重量%），可出乎意料地提供更有效的抗發炎效果，從而可以降低玻尿酸之用量，進而減輕或免除玻尿酸所引起之暫時性發炎。</p><p>此外，如後附實施例所呈現，在不避光之情形下，根據本發明所提供之抗發炎醫藥組合物中的HMG-CoA還原酶抑制劑無法在長時間儲存下維持穩定性，會有HMG-CoA還原酶抑制劑析出，不利於該組合物之實際應用。相較於此，避光儲存則可避免前述HMG-CoA還原酶抑制劑析出的問題。</p><p>由於根據本發明所提供之套組及醫藥組合物可改善習知玻尿酸製劑之缺點，故亦可應用於玻尿酸之任何已知用途，而不限於抗關節炎。舉例言之，可將本發明組合物運用於美容產品或整形外科，例如可將其添加於皮膚保養品或臉部的玻尿酸注射劑中。</p><p>於根據本發明所提供之套組及醫藥組合物中，玻尿酸與HMG-CoA還原酶抑制劑僅需簡單混合即可，故具有製程簡便、適於大規模量產之優點。</p><p>茲以下列實施例進一步例示說明本發明。其中該等實施例僅提供作為說明，而非用以限制本發明之保護範圍。本發明保護範圍係如後附申請專利範圍所示。</p><p><b>實施例</b></p><p><b>[</b><b>製備實施例</b><b>]</b><b>：製備含不同玻尿酸：</b><b>HMG-CoA</b><b>還原酶抑制劑重量配比之關節注射液</b></p><p><b>A-1.</b><b>玻尿酸：</b><b>HMG-CoA</b><b>還原酶抑制劑</b><b>= 99.5</b><b>：</b><b>0.5</b></p><p>於一預裝式玻尿酸注射針器（購自菲利達生技股份有限公司）內，添加1毫升之等張溶液，其包含0.2毫克之玻尿酸（購自菲利達生技股份有限公司；平均分子量為60至80萬道爾頓）及0.5重量%（以玻尿酸及HMG-CoA還原酶抑制劑之總重計）之HMG-CoA還原酶抑制劑（瑞舒伐史坦酊（Rosuvastatin），購自台灣丞泰企業有限公司（Forefront Enterprise Co., Ltd. Taiwan））作為主要成分，再添加5至20毫克之氯化鈉、硫酸氫鈉、及雙氫硫酸鈉，最後添加注射用水作為賦型劑至總體積為2毫升，以製得一玻尿酸關節注射液，並置於室溫下避光儲存。</p><p><b>A-2.</b><b>玻尿酸：</b><b>HMG-CoA</b><b>還原酶抑制劑</b><b>= 99</b><b>：</b><b>1</b></p><p>比照A-1進行製備，但將HMG-CoA還原酶抑制劑（瑞舒伐史坦酊）之用量改為以玻尿酸及HMG-CoA還原酶抑制劑（瑞舒伐史坦酊）之總重計為1重量%，以製得一玻尿酸關節注射液，並置於室溫下避光儲存。</p><p><b>A-3.</b><b>玻尿酸：</b><b>HMG-CoA</b><b>還原酶抑制劑</b><b>= 98.5</b><b>：</b><b>1.5</b></p><p>比照A-1進行製備，但將HMG-CoA還原酶抑制劑（瑞舒伐史坦酊）之用量改為以玻尿酸及HMG-CoA還原酶抑制劑（瑞舒伐史坦酊）之總重計為1.5重量%，以製得一玻尿酸關節注射液，並置於室溫下避光儲存。</p><p><b>A-4. 98</b><b>重量</b><b>%</b><b>玻尿酸與</b><b>2</b><b>重量</b><b>%HMG-CoA</b><b>還原酶抑制劑</b></p><p>比照A-1進行製備，但將HMG-CoA還原酶抑制劑（瑞舒伐史坦酊）之用量改為以玻尿酸及HMG-CoA還原酶抑制劑（瑞舒伐史坦酊）之總重計為2重量%，以製得一玻尿酸關節注射液，並置於室溫下避光儲存。</p><p><b>B</b><b>、纖維母細胞樣滑膜細胞之製備</b></p><p>以下述操作，於關節鏡清創手術中，收集來自四個罹患有退化性關節炎（osteoarthritis，OA）之病患的纖維母細胞樣滑膜細胞（fibroblast-like synoviocytes，FLS）並進行培養。</p><p>首先，將病患之關節滑膜（synovium）切成小碎片，再使其懸浮於一DMEM（Dulbecco modified eagle’s medium）培養基中，並於37
<sup>o</sup>C、5%之二氧化碳的環境中培養3天。該培養基含有1.5公克／公升碳酸氫鈉（S6297，Sigma-Aldrich，St Louis，密蘇里州，美國）、1%盤尼西林-鏈黴素-新黴素（P4083，Sigma-Aldrich）、以及10%胎牛血清（04-001-1A，Biological Industries，Grand Island，紐約州，美國）。
</p><p>接著，以磷酸鹽緩衝液（PBS）洗去未附著之細胞，並更換培養基，再培養留下之附著的細胞歷時2周。重複上述程序3至6次，存留下的細胞即為纖維母細胞樣滑膜細胞。</p><p><b>實施例</b><b>1</b><b>：玻尿酸關節注射液之避光試驗</b></p><p><b>（</b><b>1-1</b><b>）</b></p><p>取[製備實施例A-2]所提供之玻尿酸關節注射液，分成二組，分別盛裝於透明玻璃試管中，並於室溫下分別以如下條件保存，歷時3個月： 1. 照光組（共四管）：靜置於開燈房間中，24小時接受燈照；以及 2. 避光組（共四管）：靜置於恆溫恆濕的箱子（溫度：30
<sup>o</sup>C；相對溼度：65%）中。
</p><p>於保存期間，每月定期檢測各組注射液之流變性質G’與G＂，並取各組注射液的其中一管，以篩網進行過濾，再進行HPLC分析，以測定各組濾液中的HMG-CoA還原酶抑制劑（瑞舒伐史坦酊）含量。結果示於表1至3。</p><p>表1：流變性質（G’）
<tables><table border="1" bordercolor="#000000" width="85%"><tbody><tr><td> </td><td> 照光組 </td><td> 避光組 </td></tr><tr><td> 0個月 </td><td> 111.6 </td><td> 111.6 </td></tr><tr><td> 1個月 </td><td> 103.8 </td><td> 108.4 </td></tr><tr><td> 2個月 </td><td> 89.51 </td><td> 102.66 </td></tr><tr><td> 3個月 </td><td> 60.56 </td><td> 92.44 </td></tr></tbody></table></tables></p><p>表2：流變性質（G＂）
<tables><table border="1" bordercolor="#000000" width="85%"><tbody><tr><td> </td><td> 照光組 </td><td> 避光組 </td></tr><tr><td> 0個月 </td><td> 26.10 </td><td> 26.10 </td></tr><tr><td> 1個月 </td><td> 23.2 </td><td> 24.1 </td></tr><tr><td> 2個月 </td><td> 19.49 </td><td> 20.26 </td></tr><tr><td> 3個月 </td><td> 13.89 </td><td> 20.1 </td></tr></tbody></table></tables></p><p>表3：HMG-CoA還原酶抑制劑（瑞舒伐史坦酊）含量（mM）
<tables><table border="1" bordercolor="#000000" width="85%"><tbody><tr><td> </td><td> 照光組 </td><td> 避光組 </td></tr><tr><td> 0個月 </td><td> 0.090 </td><td> 0.090 </td></tr><tr><td> 1個月 </td><td> 0.091 </td><td> 0.096 </td></tr><tr><td> 2個月 </td><td> 0.082 </td><td> 0.098 </td></tr><tr><td> 3個月 </td><td> 0.047 </td><td> 0.089 </td></tr></tbody></table></tables></p><p><b>（</b><b>1-2</b><b>）</b>取[製備實施例A-4]所提供之玻尿酸關節注射液，比照（1-1）之方式檢測各組注射液之流變性質G’與G＂，並測定各組注射液中HMG-CoA還原酶抑制劑（瑞舒伐史坦酊）含量，以及觀察注射液之外觀變化。結果示於表4至7。
</p><p>表4：流變性質（G’）
<tables><table border="1" bordercolor="#000000" width="85%"><tbody><tr><td> </td><td> 照光組 </td><td> 避光組 </td></tr><tr><td> 0個月 </td><td> 124.1 </td><td> 124.1 </td></tr><tr><td> 1個月 </td><td> 118.7 </td><td> 120.3 </td></tr><tr><td> 2個月 </td><td> 99.59 </td><td> 118.1 </td></tr><tr><td> 3個月 </td><td> 80.32 </td><td> 106.4 </td></tr></tbody></table></tables></p><p>表5：流變性質（G＂）
<tables><table border="1" bordercolor="#000000" width="85%"><tbody><tr><td> </td><td> 照光組 </td><td> 避光組 </td></tr><tr><td> 0個月 </td><td> 26.99 </td><td> 26.99 </td></tr><tr><td> 1個月 </td><td> 22.3 </td><td> 25 </td></tr><tr><td> 2個月 </td><td> 21.06 </td><td> 23.47 </td></tr><tr><td> 3個月 </td><td> 18.46 </td><td> 23.23 </td></tr></tbody></table></tables></p><p>表6：HMG-CoA還原酶抑制劑（瑞舒伐史坦酊）含量（mM）
<tables><table border="1" bordercolor="#000000" width="85%"><tbody><tr><td> </td><td> 照光組 </td><td> 避光組 </td></tr><tr><td> 0個月 </td><td> 0.199 </td><td> 0.199 </td></tr><tr><td> 1個月 </td><td> 0.174 </td><td> 0.204 </td></tr><tr><td> 2個月 </td><td> 0.146 </td><td> 0.208 </td></tr><tr><td> 3個月 </td><td> 0.127 </td><td> 0.197 </td></tr></tbody></table></tables></p><p>表7：外觀
<tables><table border="1" bordercolor="#000000" width="85%"><tbody><tr><td> </td><td> 照光組 </td><td> 避光組 </td></tr><tr><td> 3個月 </td><td> 白色混濁 </td><td> 無色透明 </td></tr></tbody></table></tables></p><p>由表1至表7可知，經過3個月的保存，照光組玻尿酸關節注射液之流變性質及HMG-CoA還原酶抑制劑（瑞舒伐史坦酊）於其中之含量皆明顯降低，且肉眼即可觀察到該注射液有白色混濁物。然而，即使經過3個月的保存，避光組玻尿酸關節注射液之外觀、流變性質及HMG-CoA還原酶抑制劑（瑞舒伐史坦酊）於其中之含量仍無明顯變化。</p><p>上述結果顯示，於避光環境下儲存含有溶解有HMG-CoA還原酶抑制劑之高分子量玻尿酸溶液，可避免HMG-CoA還原酶抑制劑自高分子量玻尿酸中析出，有效提高該均勻溶液於架儲期內之穩定性，有利於臨床上之應用。</p><p><b>實施例</b><b>2</b><b>：玻尿酸關節注射液之細胞試驗</b></p><p><b>（</b><b>2-1</b><b>）細胞處理</b></p><p>以不含血清之培養基培養[製備實施例B]所製得之纖維母細胞樣滑膜細胞24小時後，將細胞分成五組，並分別以下述培養基進行培養，歷時24小時： （1） 控制組：含有10%胎牛血清之DMEM培養基； （2） 玻尿酸組：含有10%胎牛血清及0.1毫克／毫升玻尿酸（購自菲利達生技股份有限公司；平均分子量為60至80萬道爾頓）之DMEM培養基； （3） 0.6%RC組（玻尿酸: HMG-CoA還原酶抑制劑 = 99.4: 0.6）：含有10%胎牛血清、0.1毫克／毫升玻尿酸、及0.6重量%（以玻尿酸及瑞舒伐史坦酊之總重計）瑞舒伐史坦酊（Rosuvastatin）之DMEM培養基； （4） 1.5%RC組（玻尿酸: HMG-CoA還原酶抑制劑 = 98.5: 1.5）：含有10%胎牛血清、0.1毫克／毫升玻尿酸、及1.5重量%（以玻尿酸及瑞舒伐史坦酊之總重計）瑞舒伐史坦酊之DMEM培養基；以及 （5） 2%RC組（玻尿酸: HMG-CoA還原酶抑制劑 = 98: 2）：含有10%胎牛血清、0.1毫克／毫升玻尿酸、及2重量%（以玻尿酸及瑞舒伐史坦酊之總重計）瑞舒伐史坦酊之DMEM培養基。</p><p>接著，分別收集各組之細胞培養上清液，以進行後續試驗。</p><p><b>（</b><b>2-2</b><b>）關節炎相關因子之表現量測定</b></p><p>使用夾層結合蛋白質分析套組（sandwich binding protein assay kit或sandwich ELISA kits，購自eBioscience公司），並依據廠商之使用手冊，利用ELISA讀取器（Sunrise Remote，TECAN）測定（2-1）所收集上清液中二個與退化性關節炎相關因子的濃度，以觀察其表現程度，藉此判斷細胞之發炎情形，該二細胞激素分別為腫瘤壞死因子-α（TNF-α；標準品：88-7340，購自eBioscience）及基質金屬蛋白酶-3（MMP-3；標準品：DY513，購自R&D systems）。各組樣本皆重複進行二次試驗。最後，對所得數據進行雙樣本t檢驗（two-sample t-test），並以控制組的結果為基準（即，將控制組的表現量設定為1倍），計算各組的關節炎相關因子表現量。結果示於表8至9及圖1至2。</p><p>表8
<tables><table border="1" bordercolor="#000000" width="85%"><tbody><tr><td> </td><td> TNF-α相對表現量（倍數） </td><td> 標準差 </td></tr><tr><td> 控制組 </td><td> 1.000 </td><td> 0.000 </td></tr><tr><td> 玻尿酸組 </td><td> 0.959 </td><td> 0.061 </td></tr><tr><td> 0.6%RC組 </td><td> 0.867 </td><td> 0.118 </td></tr><tr><td> 1.5%RC組 </td><td> 0.874 </td><td> 0.101 </td></tr><tr><td> 2%RC組 </td><td> 0.945 </td><td> 0.048 </td></tr></tbody></table></tables></p><p>表9
<tables><table border="1" bordercolor="#000000" width="85%"><tbody><tr><td> </td><td> MMP-3相對表現量（倍數） </td><td> 標準差 </td></tr><tr><td> 控制組 </td><td> 1.000 </td><td> 0.000 </td></tr><tr><td> 玻尿酸組 </td><td> 0.967 </td><td> 0.073 </td></tr><tr><td> 0.6%RC組 </td><td> 0.850 </td><td> 0.117 </td></tr><tr><td> 1.5%RC組 </td><td> 0.857 </td><td> 0.118 </td></tr><tr><td> 2%RC組 </td><td> 0.950 </td><td> 0.100 </td></tr></tbody></table></tables></p><p><b>（</b><b>2-3</b><b>）試驗結果</b></p><p>由表8及圖1可知，相較於控制組，其他各組細胞之TNF-α表現量皆明顯下降，其中，玻尿酸組係下降4.1%、0.6%RC組係下降13.3%，1.5%RC組係下降12.6%，2%RC組則下降5.5%。另一方面，不論相較於玻尿酸組或2%RC組，0.6%RC組及1.5%RC組細胞之TNF-α表現量亦明顯下降。</p><p>由表9及圖2可知，相較於控制組，其他各組細胞之MMP-3表現量皆明顯下降，其中，玻尿酸組係下降3.3%、0.6%RC組係下降15%，1.5%RC組係下降14.3%，2%RC組則下降5%。另一方面，不論相較於玻尿酸組或2%RC組，0.6%RC組及1.5%RC組細胞之MMP-3表現量亦明顯下降。
</p><p>上述結果顯示，來自退化性關節炎患者之關節細胞，會分泌大量的發炎介質TNF-α及MMP-3，說明其發炎情況嚴重(如控制組所示)，單獨以玻尿酸處理後可降低前述發炎現象。另一方面，若結合HMG-CoA還原酶抑制劑與玻尿酸，則可進一步加強玻尿酸抑制發炎的效果。此外，相較於結合使用玻尿酸與2重量%(以玻尿酸與HMG-CoA還原酶抑制劑之總重計)以上之HMG-CoA還原酶抑制劑，結合使用玻尿酸與相對極少量之HMG-CoA還原酶抑制劑(例如，以玻尿酸與HMG-CoA還原酶抑制劑之總重計，HMG-CoA還原酶抑制劑含量為約0.5重量%至1.5重量%)可提供明顯較為優異之抑制發炎功效。此試驗結果益證，本發明所界定的玻尿酸與HMG-CoA還原酶抑制劑的用量的數值限定範圍具有臨界意義，本發明確實可提供無法輕易預期的有益功效。</p></mode-for-invention><description-of-drawings><description-of-element /><p>圖1及圖2係顯示含有不同HMG-CoA還原酶抑制劑：玻尿酸重量配比之注射液於抑制發炎的效益，其中，圖1顯示以控制組為基準之各組細胞培養液中的TNF-α表現量，圖2顯示以控制組為基準之各組細胞培養液中的MMP-3表現量（X代表玻尿酸；Y代表HMG-CoA還原酶抑制劑；*表示有顯著差異，p＜0.05；**表示有顯著差異，p＜0.01）。</p></description-of-drawings><bio-deposit /><sequence-list-text /><title lang="zh">Set for anti-inflammatory </title> <title lang="en">KIT FOR INHIBITING INFLAMMATION </title> <technical-field> <p>The present invention relates to a kit for anti-inflammation, which comprises (1) a first part, which is a container, has a receiving space and is protected from light; and (2) a second part, It is a homogeneous solution comprising (a) hyaluronic acid and (b) a 3-hydroxy-3-methylpentadiazine-coenzyme A (HMG-CoA) reductase inhibitor, wherein the second portion is present in the volume The space is placed in contact with the first portion. Preferably, the second portion is an anti-inflammatory pharmaceutical composition, wherein the component (a) is present in an amount of from about 98.5 wt% to 99.5% by weight based on the total weight of the component (a) and the component (b). The content of (b) is from about 0.5% by weight to 1.5% by weight. </p> </technical-field> <background-art> <p>Arthritis is a common chronic disease that causes joint pain due to degeneration of articular cartilage or inflammation of connective tissue, which in turn affects normal joint activity. Arthritis can be divided into more than one hundred types according to the site and cause. The most common ones include osteoarthritis (degenerative arthritis), rheumatoid arthritis, gouty arthritis, bacterial arthritis, and stiffness. Spinal arthritis, and lupus erythematosus. </p> <p>For the treatment of arthritis, conservative, non-surgical treatments, such as drug therapy and injection therapy, are generally used at the beginning, and surgical treatment is used when the initial treatment is not effective. Drug treatments are known to include anti-inflammatory drugs using steroids and non-steroids. Among them, although steroid drugs have the advantage of rapid and significant analgesic effects, they cause many side effects such as osteoporosis, wound healing, upper gastrointestinal bleeding, and even It will aggravate the symptoms of diseases such as high blood pressure or diabetes. As for non-steroidal drugs, although they have good analgesic effects, if used for a long time, side effects such as peptic ulcer, lower extremity edema and kidney function damage may occur. </p> <p>Hyaluric acid, also known as "hyaluronic acid" or "uronic acid", is known to have an anti-inflammatory effect, so it is widely used in injections for inhibiting osteoarthritis, for example, injection of hyaluronic acid directly into a patient. Joints to moderately relieve inflammation and pain in patients. In addition, hyaluronic acid can also act as a lubricant to help joint movements, while at the same time improving joint function. However, it has been found that hyaluronic acid can effectively alleviate pain, but when it enters the human body, it sometimes causes a temporary inflammatory reaction within a few days, and even causes chronic inflammation, which is not conducive to the anti-inflammatory benefits (see Leopold). Et al., Increased frequency of acute local reaction to intra-articular hylan GF-20 (Synvisc) in patients receiving more than one course of treatment.
<i>J Bone Joint Surg </i>, 2002; 84: 1619-23; Bernardeau et al., Acute arthritis after intra-articular hyaluronate injection: onset of effusions without crystal.
<i>Ann Rheum Dis </i>, 2001; 60: 518-20; and Kroesen et al., Induction of an acute attack of calcium pyrophosphate dihydrate arthritis by intra-articular injection of hylan G-F 20 (Synvisc).
<i>Clin Rheumatol </i>, 2000; 19: 147-9, the entire contents of which are hereby incorporated by reference. Therefore, if the desired anti-inflammatory effect can be provided at a lower hyaluronic acid dose, the subsequent inflammatory reaction possibly caused by hyaluronic acid entering the human body can be alleviated or even avoided.
</p> <p>The inventors of the present invention found that by using hyaluronic acid in combination with a relatively small amount of HMG-CoA reductase inhibitor, an unexpected anti-inflammatory effect can be achieved, and the amount of hyaluronic acid can be effectively reduced, thereby reducing or eliminating the cause of hyaluronic acid. Temporary inflammation. </p> <p>Although high molecular weight hyaluronic acid has a better anti-inflammatory effect than low molecular weight hyaluronic acid, the inventors of the present invention found that 3-hydroxy-3-methylpentadiazine coenzyme A (hereinafter referred to as "HMG-CoA" The reductase inhibitor is mixed with high molecular weight hyaluronic acid to provide a homogeneous solution, and the HMG-CoA reductase inhibitor therein is easily precipitated from the solution and cannot be stably present. Further studies have found that the above-mentioned homogeneous solution provided by mixing HMG-CoA reductase inhibitor with high molecular weight hyaluronic acid can be stably stored in a dark environment, which is beneficial to the clinical application of the solution. </p> </background-art> <disclosure> <p>An object of the present invention is to provide a kit for anti-inflammation, comprising: (1) a first portion which is a container having a receiving space and being protected from light; and (2) a The second part is a homogeneous solution comprising (a) hyaluronic acid and (b) a 3-hydroxy-3-methylpentadiazine coenzyme A (HMG-CoA) reductase inhibitor, wherein the second part is It is present in the accommodating space and is in contact with the first portion. The average molecular weight of component (a) in the second portion is preferably from about 300,000 to 6 million Daltons, more preferably from about 2 million to 6 million dolphins. Preferably, component (b) in the second portion is selected from at least one of the group consisting of: Atorvastatin, Cerivastatin, Fluvastatin (Fluvastatin), Levastatin, Mevastatin, Pitavastatin, Pravastatin, Simvastatin, And Rosuvastatin. </p> <p> Another object of the present invention is to provide a pharmaceutical composition comprising (a) hyaluronic acid, and (b) a 3-hydroxy-3-methylpentadiazine-CoA reductase A (HMG-CoA) reductase The inhibitor, wherein the content of the component (a) is from about 98.5 wt% to 99.5% by weight based on the total weight of the component (a) and the component (b), and the content of the component (b) is from about 0.5 wt% to 1.5 wt. %. Wherein, the average molecular weight of the component (a) is preferably from about 300,000 to 6 million Daltons, more preferably from about 2 million to 6 million dolphins; and the component (b) is preferably selected from the group below. At least one of them: Atorvastatin, Cerivastatin, Fluvastatin, Lovastatin, Mevastatin ), Pitavastatin, Pravastatin, Simvastatin, and Rosuvastatin. The foregoing pharmaceutical compositions may be used as the second part of the kit of the invention, and may further comprise (c) a pharmaceutically acceptable carrier, if desired. </p> The detailed technical content and some of the specific embodiments of the present invention will be described in the following, and the features of the present invention will be apparent to those of ordinary skill in the art. </p> </disclosure> <mode-for-invention> The detailed description of the present invention and the preferred embodiments of the present invention will be described in the following description of the present invention. The invention may be practiced in a variety of different forms, and the scope of the invention should not be construed as being limited by the description. In addition, the terms "a", "an" and "the" </p> <p>Hyaluronic acid is a glycosaminoglycan that does not contain sulfur. Its basic structure is a large polysaccharide composed of two disaccharide units D-glucuronic acid and N-acetylglucosamine, which has the following formula (I ) chemical formula:
<img he="110" wi="222" img-format="jpg" id="i0002" img-content="drawing" orientation="portrait" inline="no" file="TWI663979B_D0001.tif" /> (I).
</p> <p>Hyaluronic acid is one of the main components of the extracellular matrix. It is widely distributed in endothelial tissues, connective tissues, epidermal tissues and nervous tissues. It is essential for physiological activities such as cell proliferation and migration, and is within the dermis of the skin. It is an important moisturizing ingredient with excellent adhesiveness and stretchability. It is an ideal filling material and is often used in beauty products and plastic surgery. The currently commercially available hyaluronic acid solution (for example, a preparation containing an aqueous solution of sodium hyaluronate) is contained in a transparent container, and the storage period at normal temperature is three years. </p> <p>The inventors of the present invention found that storing a high molecular weight hyaluronic acid solution containing a HMG-CoA reductase inhibitor in a dark environment can prevent the precipitation of HMG-CoA reductase inhibitor from high molecular weight hyaluronic acid, thereby effectively improving the uniformity. The stability of the solution during the shelf life is beneficial to clinical applications. Accordingly, the present invention provides a kit for anti-inflammatory, comprising: (1) a first portion which is a container having a housing space and protected from light; and (2) a second portion A homogeneous solution comprising (a) hyaluronic acid and (b) an HMG-CoA reductase inhibitor, wherein the second portion is present in the accommodating space and is in contact with the first portion. </p> <p> In the kit according to the present invention, the container bottle, can, or bag as the first portion may be provided in any suitable material without particular limitation. </p> <p>About the light-shielding container as the first part, the term "protectable from light" means that light having a wavelength of 290 to 450 nanometers (nm) can be shielded to make the transmittance of any wavelength in the wavelength range. Not more than 10%. That is, the light-shielding container is shielded by at least 90% for light having a wavelength of any of 290 to 450 nanometers (nm). Suitable materials for providing the light-resistant container can be found, for example, in the United States Pharmacopeial Convention.
<i>United States Pharmacopeia 29 </i>, United States Pharmacopeial Convention, Rockville, Md, USA, 2012), the aforementioned Pharmacopoeia is hereby incorporated by reference. For example, but not limited thereto, Type I glass, Type II glass, Type III glass, Type NP glass, or Plastic classes I-VI provide the light-proof container as the first part.
</p> <p> In the kit according to the present invention, the hyaluronic acid as the component (a) of the second part preferably has an average molecular weight of about 300,000 to 6 million Daltons, more preferably an average molecular weight of about 2 million. Up to 6 million Daltons. </p> <p>A hyaluronic acid suitable for use in a kit according to the present invention may be prepared from natural materials (for example, extracted from living parts such as chicken crown, umbilical cord, cartilage, and skin), or may be chemically synthesized or utilized by microorganisms such as yeast. Culture or genetic engineering methods of production. Commercially available hyaluronic acid preparations may also be used to provide kits according to the invention, such as formulations containing aqueous sodium hyaluronate, including ARTZ Dispo
<sup>® </sup>, SYNVISC
<sup>® </sup>, HYALGAN
<sup>® </sup>, and ORTHOVISC
<sup>® </sup>, or a formulation comprising an aqueous solution of cross-linked hyaluronic acid derivative, including SYNVISC
<sup>® </sup> and DUROLANE
<sup>® </sup>.
</p> <p> The hyaluronic acid suitable for the kit according to the present invention may be in a free state in which no salt is formed, and a pharmaceutically acceptable salt may also be formed. The pharmaceutically acceptable salt of hyaluronic acid includes a salt formed with an inorganic base, such as an alkali metal ion salt such as a sodium salt or a potassium salt, an alkaline earth metal ion salt such as a magnesium salt or a calcium salt, and an ammonium salt; or A salt formed, for example, a salt formed with diethanolamine, cyclohexylamine or an amino acid. </p> <p> In the kit according to the present invention, the component (b) as the second component is an HMG-CoA reductase inhibitor, which includes a class of drugs called "statins", which The statins include the compounds shown in the following table:
<tables> <table border="1" bordercolor="#000000" width="85%"> <tbody> <tr> <td> name </td> <td> chemical formula </td> <td> Product Name </td> </tr> <tr> <td> Atostatin (Atorvastatin) </td> <td> <img wi="216" he="110" file="02_image004.tif" img-format="tif"/> </img> </td> <td> Lipitor or Torvast </td> </tr> <tr> <td> Cerivastatin </td> <td> <img wi="199" he="117" file="02_image006.tif" img-format="tif"/> </img> </td> <td> Lipobay or Baycol </td> </tr> <tr> <td> Fluvastatin </td> <td> <img wi="155" he="163" file="02_image008.tif" img-format="tif"/> </img> </td> <td> Lescol or Lescol XL </td> </tr> <tr> <td> Lovastatin </td> <td> <img wi="170" he="151" file="02_image010.tif" img-format="tif"/> </img> </td> <td> Mevacor, Altocor or Altoprev </td> </tr> <tr> <td> Mevastatin </td> <td> <img wi="149" he="151" file="02_image012.tif" img-format="tif"/> </img> </td> <td> none </td> </tr> <tr> <td> Pitavastatin </td> <td> <img wi="193" he="121" file="02_image014.tif" img-format="tif"/> </img> </td> <td> Livalo or Pitava </td> </tr> <tr> <td> Pravastatin </td> <td> <img wi="185" he="155" file="02_image016.tif" img-format="tif"/> </img> </td> <td> Pravachol, Selektine or Lipostat </td> </tr> <tr> <td> Simvastatin </td> <td> <img wi="144" he="136" file="02_image018.tif" img-format="tif"/> </img> </td> <td> Zocor or Lipex </td> </tr> <tr> <td> Rosuvastatin </td> <td> <img wi="222" he="121" file="02_image020.tif" img-format="tif"/> </img> </td> <td> Crestor </td> </tr> </tbody> </table> </tables> </p> <p> Thus, in some preferred embodiments of the kit according to the invention, component (b) as the second component is selected from at least one of the following groups: Atorvastatin , Cerivastatin, Fluvastatin, Lovastatin, Mevastatin, Pitavastatin, Devastatation Pravastatin, Simvastatin, and Rosuvastatin. </p> <p> The present invention further provides an anti-inflammatory pharmaceutical composition comprising (a) hyaluronic acid and (b) an HMG-CoA reductase inhibitor, wherein the hyaluronic acid and the HMG-CoA reductase inhibitor are as described above. The pharmaceutical composition can be used as the second part of the kit provided in accordance with the present invention and can be in any convenient form without particular limitation. By way of example, but not by way of limitation, the pharmaceutical composition may be provided in the form of subcutaneous, intravenous, or intra-articular injection. Wherein, depending on the form of use and use, the pharmaceutical composition may further comprise a pharmaceutically acceptable carrier, wherein the carrier comprises an excipient, a diluent, an adjuvant, a stabilizer, an absorption delaying agent, a disintegrating agent, Solubilizers, emulsifiers, antioxidants, binders, binders, tackifiers, dispersants, suspending agents, lubricants, hygroscopic agents, and the like. </p> <p> exemplified by injections or drips suitable for subcutaneous injection, intravenous injection, or intra-articular injection, one or more, for example, isotonic solutions, salt buffers, may be included in the pharmaceutical compositions provided according to the present invention ( Ingredients such as phosphate buffer or citrate buffer, solubilizer, emulsifier, 5% sugar solution, and other carriers, intravenous infusion, emulsion intravenous infusion, dry powder injection, suspension injection, or dry powder A pharmaceutical composition such as a suspension injection or the like is provided. Alternatively, the pharmaceutical composition can be prepared as a pre-injection solid, the pre-injection solid can be provided in a dosage form, or emulsifiable, which is soluble in other solutions or suspensions, and prior to administration to an individual in need thereof, The solid before dissolution is dissolved in other solutions or suspensions or emulsified to provide the desired injection. </p> <p> Optionally, a suitable amount of an additive such as a buffer, a preservative, a preservative, an antibacterial agent, an antifungal agent or the like may be further contained in the anti-inflammatory pharmaceutical composition provided according to the present invention. In addition, one or more other active ingredients may be further included in the pharmaceutical composition as needed to further enhance the efficacy or increase the flexibility and blending of the pharmaceutical composition, as long as the other active ingredients are beneficial to the pharmaceutical composition of the present invention. There is no adverse effect. Examples of such other active ingredients include, but are not limited to, steroid anti-inflammatory drugs, non-steroidal anti-inflammatory drugs, glucosamine, and the like. </p> <p> In the anti-inflammatory pharmaceutical composition provided according to the present invention, the ratio of the component (a) to the component (b) is not particularly limited. In general, the content of the component (a) is from about 80% by weight to 99.9% by weight based on the total weight of the component (a) and the component (b), and the content of the component (b) is from about 0.1% by weight to 20% by weight. %. Preferably, the content of the component (a) is from about 98% by weight to 99.9% by weight based on the total weight of the component (a) and the component (b), and the content of the component (b) is from about 0.1% by weight to 2% by weight. %. More preferably, the content of the component (a) is from about 98.5 wt% to 99.5% by weight based on the total weight of the component (a) and the component (b), and the content of the component (b) is from about 0.5 wt% to 1.5 wt. %. </p> <p> Since the pharmaceutical composition of the present invention can provide an enhanced anti-inflammatory effect, it can be used for inhibiting inflammation, and particularly for inhibiting arthritis, including osteoarthritis (degenerative arthritis), rheumatoid arthritis, gouty joints Inflammation, bacterial arthritis, ankylosing spondylarthritis, and lupus erythematosus are preferred for inhibiting osteoarthritis and rheumatoid arthritis. In one embodiment of the present invention, the pharmaceutical composition of the present invention is formulated into an injection for use in joint injection for the treatment of arthritis. </p> <p>Viewing the needs, age, weight, and health condition of the individual, the anti-inflammatory drug combination according to the present invention may be administered at different administration frequencies, such as once a day, multiple times a day, or several times a day. The application is to an individual in need thereof. In the pharmaceutical composition provided according to the present invention, the content ratio of the component (a) to the component (b) can be adjusted depending on the actual application requirements. </p> <p> As shown in the appended Examples, the inventors have found that by using hyaluronic acid in combination with a relatively small amount of HMG-CoA reductase inhibitor (for example, based on the total weight of hyaluronic acid and HMG-CoA reductase inhibitor, A content of from about 0.5% to about 1.5% by weight, which unexpectedly provides a more effective anti-inflammatory effect, can reduce the amount of hyaluronic acid, thereby reducing or eliminating temporary inflammation caused by hyaluronic acid. </p> <p> Further, as shown in the appended Examples, the HMG-CoA reductase inhibitor in the anti-inflammatory pharmaceutical composition provided according to the present invention cannot maintain stability under long-term storage without being protected from light. There will be precipitation of HMG-CoA reductase inhibitor, which is not conducive to the practical application of the composition. In contrast, the light-proof storage avoids the problem of precipitation of the aforementioned HMG-CoA reductase inhibitor. </p> <p> Since the kits and pharmaceutical compositions provided according to the present invention can improve the disadvantages of conventional hyaluronic acid preparations, they can also be applied to any known use of hyaluronic acid, and are not limited to anti-arthritis. For example, the composition of the present invention can be applied to a cosmetic product or orthopedics, for example, it can be added to a skin care product or a hyaluronic acid injection of the face. </p> <p> In the kit and the pharmaceutical composition provided by the present invention, hyaluronic acid and the HMG-CoA reductase inhibitor need only be simply mixed, so that the process is simple and suitable for mass production. </p> <p> The invention is further illustrated by the following examples. The embodiments are provided by way of illustration only and are not intended to limit the scope of the invention. The scope of the invention is shown in the appended claims. </p> <p> <b>Example </b> </p> <p> <b>[ </b> <b>Preparation Example </b> <b>] </b> <b>: Preparation with different hyaluronic acid: </b> <b>HMG-CoA </b> <b>Reductase inhibitor weight ratio joint injection </b> </p> <p> <b>A-1. </b> <b>Hyaluronic acid: </b> <b>HMG-CoA </b> <b>Reductase inhibitor </b> <b>= 99.5 </b> <b>: </b> <b>0.5 </b> </p> <p>In a preloaded hyaluronic acid injection needle (purchased from Felida Biotech Co., Ltd.), a 1 ml isotonic solution containing 0.2 mg of hyaluronic acid (purchased from Felida Biotech Co., Ltd.; An average molecular weight of 60 to 800,000 Daltons and 0.5% by weight (based on the total weight of hyaluronic acid and HMG-CoA reductase inhibitors) of HMG-CoA reductase inhibitor (Rosuvastatin, Purchased from Forefront Enterprise Co., Ltd. (Taiwan) as the main ingredient, adding 5 to 20 mg of sodium chloride, sodium hydrogen sulfate, and sodium hydrogen sulfate, and finally adding water for injection. The formulation was applied to a total volume of 2 ml to prepare a hyaluronic acid joint injection and stored at room temperature in the dark. </p> <p> <b>A-2. </b> <b>Hyaluronic acid: </b> <b>HMG-CoA </b> <b>Reductase inhibitor </b> <b>= 99 </b> <b>: </b> <b>1 </b> </p> <p>Prepare to A-1, but change the amount of HMG-CoA reductase inhibitor (Rushurastatin) to hyaluronic acid and HMG-CoA reductase inhibitor (Rushurastatin) The total weight was 1% by weight to prepare a hyaluronic acid joint injection and stored at room temperature in the dark. </p> <p> <b>A-3. </b> <b>Hyaluronic acid: </b> <b>HMG-CoA </b> <b>Reductase inhibitor </b> <b>= 98.5 </b> <b>: </b> <b>1.5 </b> </p> <p>Prepare to A-1, but change the amount of HMG-CoA reductase inhibitor (Rushurastatin) to hyaluronic acid and HMG-CoA reductase inhibitor (Rushurastatin) The total weight was 1.5% by weight to prepare a hyaluronic acid joint injection and stored at room temperature in the dark. </p> <p> <b>A-4. 98 </b> <b>weight </b> <b>% </b> <b>Hyaluronic acid and </b> <b>2 </b> <b>weight </b> <b>%HMG-CoA </b> <b>Reductase inhibitor </b> </p> <p>Prepare to A-1, but change the amount of HMG-CoA reductase inhibitor (Rushurastatin) to hyaluronic acid and HMG-CoA reductase inhibitor (Rushurastatin) The total weight was 2% by weight to prepare a hyaluronic acid joint injection and stored at room temperature in the dark. </p> <p> <b>B </b> <b> Preparation of fibroblast-like synoviocytes </b> </p> <p>In the arthroscopic debridement operation, fibroblast-like synoviocytes (FLS) from four patients with degenerative arthritis (OA) were collected and operated as follows. Cultivate. </p> <p>First, the synovium of the patient's synovium is cut into small pieces, which are then suspended in a DMEM (Dulbecco modified eagle's medium) medium, and at 37
<sup>o </sup>C, 5% carbon dioxide in an environment for 3 days. The medium contained 1.5 g/L sodium bicarbonate (S6297, Sigma-Aldrich, St Louis, Missouri, USA), 1% penicillin-streptomycin-neomycin (P4083, Sigma-Aldrich), and 10% fetal calf Serum (04-001-1A, Biological Industries, Grand Island, New York, USA).
</p> <p> Next, unattached cells were washed away with phosphate buffered saline (PBS), and the medium was replaced, and the remaining attached cells were cultured for 2 weeks. The above procedure was repeated 3 to 6 times, and the remaining cells were fibroblast-like synoviocytes. </p> <p> <b>Example </b> <b>1 </b> <b>: Light-proof test of hyaluronic acid joint injection </b> </p> <p> <b>( </b> <b>1-1 </b> <b>) </b> </p> <p> The hyaluronic acid joint injection provided in [Preparation Example A-2] was divided into two groups, which were respectively placed in a transparent glass test tube, and stored at room temperature under the following conditions for 3 months: 1. Illumination group (four tubes in total): statically placed in the room with lights, 24 hours to receive the light; and 2. dark group (four tubes): static and constant humidity box (temperature: 30
<sup>o </sup>C; relative humidity: 65%).
</p> <p> During the storage period, the rheological properties G' and G" of each group of injections were periodically and monthly, and one of the injection groups of each group was taken, filtered through a sieve, and then analyzed by HPLC to determine each The content of HMG-CoA reductase inhibitor (Rushurastatin) in the filtrate was shown in Tables 1 to 3. </p> <p>Table 1: Rheological properties (G')
<tables> <table border="1" bordercolor="#000000" width="85%"> <tbody> <tr> <td> </td> <td> Photo group </td> <td> Light protection group </td> </tr> <tr> <td> 0 months </td> <td> 111.6 </td> <td> 111.6 </td> </tr> <tr> <td> 1 month </td> <td> 103.8 </td> <td> 108.4 </td> </tr> <tr> <td> 2 months </td> <td> 89.51 </td> <td> 102.66 </td> </tr> <tr> <td> 3 months </td> <td> 60.56 </td> <td> 92.44 </td> </tr> </tbody> </table> </tables> </p> <p>Table 2: Rheological properties (G")
<tables> <table border="1" bordercolor="#000000" width="85%"> <tbody> <tr> <td> </td> <td> Photo group </td> <td> Light protection group </td> </tr> <tr> <td> 0 months </td> <td> 26.10 </td> <td> 26.10 </td> </tr> <tr> <td> 1 month </td> <td> 23.2 </td> <td> 24.1 </td> </tr> <tr> <td> 2 months </td> <td> 19.49 </td> <td> 20.26 </td> </tr> <tr> <td> 3 months </td> <td> 13.89 </td> <td> 20.1 </td> </tr> </tbody> </table> </tables> </p> <p>Table 3: HMG-CoA reductase inhibitor (Rushurastatin) content (mM)
<tables> <table border="1" bordercolor="#000000" width="85%"> <tbody> <tr> <td> </td> <td> Photo group </td> <td> Light protection group </td> </tr> <tr> <td> 0 months </td> <td> 0.090 </td> <td> 0.090 </td> </tr> <tr> <td> 1 month </td> <td> 0.091 </td> <td> 0.096 </td> </tr> <tr> <td> 2 months </td> <td> 0.082 </td> <td> 0.098 </td> </tr> <tr> <td> 3 months </td> <td> 0.047 </td> <td> 0.089 </td> </tr> </tbody> </table> </tables> </p> <p> <b>( </b> <b>1-2 </b> <b>) </b> Taking the hyaluronic acid joint injection provided in [Preparation Example A-4], the rheological properties G' and G" of each group of injections were measured according to the method of (1-1), and each group of injections was measured. The content of HMG-CoA reductase inhibitor (Rushurastatin) was observed, and the appearance change of the injection was observed. The results are shown in Tables 4 to 7.
</p> <p>Table 4: Rheological properties (G')
<tables> <table border="1" bordercolor="#000000" width="85%"> <tbody> <tr> <td> </td> <td> Photo group </td> <td> Light protection group </td> </tr> <tr> <td> 0 months </td> <td> 124.1 </td> <td> 124.1 </td> </tr> <tr> <td> 1 month </td> <td> 118.7 </td> <td> 120.3 </td> </tr> <tr> <td> 2 months </td> <td> 99.59 </td> <td> 118.1 </td> </tr> <tr> <td> 3 months </td> <td> 80.32 </td> <td> 106.4 </td> </tr> </tbody> </table> </tables> </p> <p>Table 5: Rheological properties (G")
<tables> <table border="1" bordercolor="#000000" width="85%"> <tbody> <tr> <td> </td> <td> Photo group </td> <td> Light protection group </td> </tr> <tr> <td> 0 months </td> <td> 26.99 </td> <td> 26.99 </td> </tr> <tr> <td> 1 month </td> <td> 22.3 </td> <td> 25 </td> </tr> <tr> <td> 2 months </td> <td> 21.06 </td> <td> 23.47 </td> </tr> <tr> <td> 3 months </td> <td> 18.46 </td> <td> 23.23 </td> </tr> </tbody> </table> </tables> </p> <p>Table 6: HMG-CoA reductase inhibitor (Rushurastatin) content (mM)
<tables> <table border="1" bordercolor="#000000" width="85%"> <tbody> <tr> <td> </td> <td> Photo group </td> <td> Light protection group </td> </tr> <tr> <td> 0 months </td> <td> 0.199 </td> <td> 0.199 </td> </tr> <tr> <td> 1 month </td> <td> 0.174 </td> <td> 0.204 </td> </tr> <tr> <td> 2 months </td> <td> 0.146 </td> <td> 0.208 </td> </tr> <tr> <td> 3 months </td> <td> 0.127 </td> <td> 0.197 </td> </tr> </tbody> </table> </tables> </p> <p>Table 7: Appearance
<tables> <table border="1" bordercolor="#000000" width="85%"> <tbody> <tr> <td> </td> <td> Photo group </td> <td> Light protection group </td> </tr> <tr> <td> 3 months </td> <td> white turbidity </td> <td> colorless and transparent </td> </tr> </tbody> </table> </tables> </p> <p>It can be seen from Table 1 to Table 7 that after 3 months of preservation, the rheological properties of the chlorouric acid joint injection and the HMG-CoA reductase inhibitor (Rushurastatin) in the illuminating group are obvious. It was lowered and the injection was observed to have a white turbidity. However, even after 3 months of preservation, the appearance, rheological properties of the hyaluronic acid joint injection and the HMG-CoA reductase inhibitor (Rushurastatin) in the dark group did not change significantly. </p> <p>The above results show that storing a high molecular weight hyaluronic acid solution containing HMG-CoA reductase inhibitor in a dark environment can prevent the precipitation of HMG-CoA reductase inhibitor from high molecular weight hyaluronic acid, effectively improving the uniform solution. The stability during the shelf life is beneficial to clinical applications. </p> <p> <b>Example </b> <b>2 </b> <b>: Cellular test of hyaluronic acid joint injection </b> </p> <p> <b>( </b> <b>2-1 </b> <b>) Cell processing </b> </p> <p> The fibroblast-like synoviocytes prepared in [Sample Example B] were cultured in a serum-free medium for 24 hours, and the cells were divided into five groups and cultured in the following medium for 24 hours: 1) Control group: DMEM medium containing 10% fetal bovine serum; (2) Hyaluronic acid group: containing 10% fetal bovine serum and 0.1 mg/ml hyaluronic acid (purchased from Felida Biotech Co., Ltd.; average molecular weight 60 to 80 DMEM medium of Wandalton; (3) 0.6% RC group (hyaluronic acid: HMG-CoA reductase inhibitor = 99.4: 0.6): containing 10% fetal bovine serum, 0.1 mg/ml hyaluronic acid, and 0.6% by weight ( Rosuvastatin DMEM medium based on the total weight of hyaluronic acid and rosuvhrastir; (4) 1.5% RC group (hyaluronic acid: HMG-CoA reductase inhibitor = 98.5: 1.5): DMEM medium containing 10% fetal bovine serum, 0.1 mg/ml hyaluronic acid, and 1.5% by weight (based on the total weight of hyaluronic acid and rosuvhrastir) rosuvastristan; and (5) 2% RC group (Hyalouric acid: HMG-CoA reductase inhibitor = 98: 2 ): DMEM medium containing 10% fetal bovine serum, 0.1 mg/ml hyaluronic acid, and 2% by weight (based on the total weight of hyaluronic acid and rosuvhrastir) rosuvastristan. </p> <p> Next, the cell culture supernatants of the respective groups were separately collected for subsequent experiments. </p> <p> <b>( </b> <b>2-2 </b> <b>) Determination of the amount of arthritis related factors </b> </p> <p>A sandwich binding protein assay kit or a sandwich ELISA kits (available from eBioscience) was used and assayed using an ELISA reader (Sunrise Remote, TECAN) according to the manufacturer's manual (2-1) The concentration of two factors associated with degenerative arthritis in the supernatant collected to observe the degree of expression, thereby determining the inflammatory condition of the cell, which is tumor necrosis factor-α (TNF-α; standard Products: 88-7340, purchased from eBioscience) and matrix metalloproteinase-3 (MMP-3; standard: DY513, available from R&D systems). Each group of samples was repeated for a second test. Finally, a two-sample t-test was performed on the obtained data, and the arthritis-related factors of each group were calculated based on the results of the control group (ie, the expression of the control group was set to 1 time). Performance. The results are shown in Tables 8 to 9 and Figures 1 to 2. </p> <p>Table 8
<tables> <table border="1" bordercolor="#000000" width="85%"> <tbody> <tr> <td> </td> <td> TNF-α relative performance (multiple) </td> <td> standard deviation </td> </tr> <tr> <td> control group </td> <td> 1.000 </td> <td> 0.000 </td> </tr> <tr> <td> Hyaluronic acid group </td> <td> 0.959 </td> <td> 0.061 </td> </tr> <tr> <td> 0.6% RC group </td> <td> 0.867 </td> <td> 0.118 </td> </tr> <tr> <td> 1.5% RC group </td> <td> 0.874 </td> <td> 0.101 </td> </tr> <tr> <td> 2%RC group </td> <td> 0.945 </td> <td> 0.048 </td> </tr> </tbody> </table> </tables> </p> <p>Table 9
<tables> <table border="1" bordercolor="#000000" width="85%"> <tbody> <tr> <td> </td> <td> MMP-3 relative performance (multiple) </td> <td> standard deviation </td> </tr> <tr> <td> control group </td> <td> 1.000 </td> <td> 0.000 </td> </tr> <tr> <td> Hyaluronic acid group </td> <td> 0.967 </td> <td> 0.073 </td> </tr> <tr> <td> 0.6% RC group </td> <td> 0.850 </td> <td> 0.117 </td> </tr> <tr> <td> 1.5% RC group </td> <td> 0.857 </td> <td> 0.118 </td> </tr> <tr> <td> 2%RC group </td> <td> 0.950 </td> <td> 0.100 </td> </tr> </tbody> </table> </tables> </p> <p> <b>( </b> <b>2-3 </b> <b>) Test results </b> </p> <p> As can be seen from Table 8 and Figure 1, the TNF-α expression levels of the other groups were significantly lower than those of the control group, among which, the hyaluronic acid group decreased by 4.1%, and the 0.6% RC group decreased by 13.3%, 1.5. The %RC group decreased by 12.6%, while the 2% RC group decreased by 5.5%. On the other hand, TNF-α expression was significantly decreased in the 0.6% RC group and 1.5% RC group, regardless of the hyaluronic acid group or the 2% RC group. </p> <p> As can be seen from Table 9 and Figure 2, compared with the control group, the MMP-3 expression of the other groups of cells decreased significantly, among which, the hyaluronic acid group decreased by 3.3%, and the 0.6% RC group decreased by 15%. The %RC group decreased by 14.3%, and the 2% RC group decreased by 5%. On the other hand, regardless of the hyaluronic acid group or the 2% RC group, the MMP-3 expression of the cells in the 0.6% RC group and the 1.5% RC group was also significantly decreased.
</p> <p>The above results show that the joint cells from patients with degenerative arthritis secrete a large amount of inflammatory mediators TNF-α and MMP-3, indicating that the inflammation is serious (as shown in the control group), after treatment with hyaluronic acid alone Reduce the aforementioned inflammation. On the other hand, when the HMG-CoA reductase inhibitor and hyaluronic acid are combined, the effect of hyaluronic acid on inflammation can be further enhanced. In addition, hyaluronic acid was used in combination with a relatively small amount of HMG-CoA reduction compared to the combination of hyaluronic acid and 2% by weight (based on the total weight of hyaluronic acid and HMG-CoA reductase inhibitor) of HMG-CoA reductase inhibitor. The enzyme inhibitor (for example, the HMG-CoA reductase inhibitor content of from about 0.5% to about 1.5% by weight based on the total weight of the hyaluronic acid and the HMG-CoA reductase inhibitor) provides a significantly superior anti-inflammatory effect. The results of this test envisage that the numerically defined range of amounts of hyaluronic acid and HMG-CoA reductase inhibitors as defined by the present invention is of critical importance, and the present invention does provide beneficial effects that are not readily expected. </p> </mode-for-invention> <description-of-drawings> <description-of-element /> <p>Fig. 1 and Fig. 2 show the effects of different HMG-CoA reductase inhibitors: hyaluronic acid weight ratio injections in inhibiting inflammation, wherein Fig. 1 shows the cell culture medium in each group based on the control group. The amount of TNF-α expression, Figure 2 shows the amount of MMP-3 in each group of cell culture media based on the control group (X stands for hyaluronic acid; Y stands for HMG-CoA reductase inhibitor; * indicates significant difference, p <0.05; ** indicates a significant difference, p < 0.01). </p> </description-of-drawings> <bio-deposit /> <sequence-list-text />