TW202200164A - Multi-mineral supplement for improved bone density - Google Patents
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本發明之技術領域係為提供用於改善骨密度及／或骨生成的礦物質補充劑，特別是含有兩種或多種礦物質的礦物質補充劑。The technical field of the present invention is to provide mineral supplements for improving bone density and/or osteogenesis, particularly mineral supplements containing two or more minerals.
與骨骼生長及發育相關的人體疾病，例如骨質疏鬆症（osteoporosis），隨著人口老齡化，對醫療保健成本造成越來越大的負擔。雖然目前已發展出一些藥物來治療這些病症，但是這些醫療方式可能仍具有缺點。例如，荷爾蒙補充療法（hormone replacement therapy）雖然可以有效預防更年期婦女骨質疏鬆症的發展，但可能導致某些癌症發展的風險增加。雖然目前已經開發了更有標靶性的化合物（例如，抑制骨吸收（bone resorption）的雙膦酸酯類（bisphosphonates）），但其與髖骨及股骨骨折有相關性。因此，有一些研究人員已經開始尋找更簡單的方法，例如補充礦物質。Human diseases related to bone growth and development, such as osteoporosis, are placing an increasing burden on healthcare costs as the population ages. Although some drugs have been developed to treat these conditions, these medical modalities may still have drawbacks. For example, hormone replacement therapy, while effective at preventing the development of osteoporosis in menopausal women, may lead to an increased risk of developing certain cancers. Although more targeted compounds (eg, bisphosphonates that inhibit bone resorption) have been developed, they have been associated with hip and femoral fractures. Therefore, some researchers have begun to look for simpler methods, such as mineral supplementation.
迄今為止，補充鈣質是用於骨密度流失的最常見的治療方法。然而，補充鈣質雖然已被證明是有用且成功的，但其高度依賴於個體的鈣吸收及利用，這反過來可能需要另外補充維生素、荷爾蒙及其他化合物。因此，單獨補充鈣質在治療與骨成長及吸收相關的病症時，其效果有限。Calcium supplementation is by far the most common treatment for bone density loss. However, calcium supplementation, although proven useful and successful, is highly dependent on the individual's calcium absorption and utilization, which in turn may require additional vitamin, hormonal, and other compound supplementation. Therefore, calcium supplementation alone has limited efficacy in treating conditions related to bone growth and resorption.
一些研究人員已經研究了補充各種有機化合物來治療這些疾病，其中一些有機化合物係與鈣以外的金屬形成複合物。例如，Yamaguchi所提出的美國專利案5,294,634描述了與鋅複合的肌肽（carnosine）用於促進骨生成的用途。本文所載之所有公開文獻皆通過引用而納為本文揭露之一部分，如同每一個別的公開文獻或專利申請案係具體地及個別地被指示藉由引用而納入本文揭露之一部分一樣。凡參考文獻中術語的定義或用法與本文所提供的該術語的定義不一致或相反時，該術語的定義係以本文所提供者為準而不適用參考文獻中的定義。Yamaguchi所提出的美國專利案5,935,996也描述了與鋅複合的異黃酮（isoflavone）用於促進骨生成，而Yamaguchi所提出的美國專利申請公開案2008/0113038描述了用於此目的之與鋅複合的β-隱黃質（beta-cryptoxanthin）的用途。類似地，Hansen等人所提出的美國專利申請公開案2010/0048697描述了麩胺酸鍶鹽（strontium salts of glutamate）及α-酮戊二酸（alpha-ketoglutarate）用於治療骨質疏鬆症的用途。Cornish及Reid所提出的國際專利申請公開案WO 2008/147228描述了將羧酸及羧酸衍生物的各種金屬鹽添加於某些乳製品中，用於治療與骨吸收或成骨細胞增生相關的病症的用途。然而，這種有機複合物的製備複雜且可能具有穩定性的問題。Some researchers have investigated the supplementation of various organic compounds, some of which form complexes with metals other than calcium, to treat these diseases. For example, US Patent No. 5,294,634 to Yamaguchi describes the use of carnosine complexed with zinc to promote osteogenesis. All publications contained herein are incorporated by reference into a part of this disclosure, as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference into a part of this disclosure. To the extent that the definition or usage of a term in a reference is inconsistent with or contrary to the definition of that term provided herein, the definition of that term is provided herein and does not apply to the definition in the reference. US Patent No. 5,935,996 to Yamaguchi also describes isoflavones complexed with zinc for promoting osteogenesis, and US Patent Application Publication 2008/0113038 to Yamaguchi describes isoflavones complexed with zinc for this purpose. Uses of beta-cryptoxanthin. Similarly, US Patent Application Publication 2010/0048697 by Hansen et al. describes the use of strontium salts of glutamate and alpha-ketoglutarate for the treatment of osteoporosis . International Patent Application Publication WO 2008/147228 by Cornish and Reid describes the addition of various metal salts of carboxylic acids and carboxylic acid derivatives to certain dairy products for the treatment of diseases associated with bone resorption or osteoblast proliferation Disease use. However, the preparation of such organic complexes is complicated and may have stability issues.
目前已經研究嘗試以使用包含鈣以外的金屬的裝置或組合物來治療與骨生長相關的病症。例如，Aghion等人所提出的美國專利申請公開案2009/0081313描述了主要由元素鎂製成的植入物用於骨科的用途。然而，鑑於已知元素鎂與水的反應性，因此，尚不清楚這種方法是否可以以可用的形式或受控的方式提供鎂。類似地，Johns等人所提出的美國專利申請公開案2012/0141599描述了使用包括金屬負載沸石（metal-loaded zeolites）的植入物用於骨移植，特別是使用了引用鋅及銅的組合。在相關方法中，Hass所提出的美國專利申請公開案2012/0141429描述了使用氧化鋅塗佈的納米結構來增強骨增生的用途。Barralet所提出的國際專利申請公開案WO 2011/058443雖然不直接指出植入物，但是描述了在需要增強骨礦化（mineralization）的部位施用各種不溶性金屬磷酸鹽類。然而，不清楚的是，是否這樣的方法對所供給的金屬用量提供足夠控制，以避免釋放有毒及／或造成細胞毒性的金屬含量。There have been research attempts to use devices or compositions containing metals other than calcium to treat conditions associated with bone growth. For example, US Patent Application Publication 2009/0081313 by Aghion et al. describes the use of implants made primarily of elemental magnesium for orthopaedics. However, given the known reactivity of elemental magnesium with water, it is unclear whether this method can provide magnesium in a usable form or in a controlled manner. Similarly, US Patent Application Publication 2012/0141599 by Johns et al. describes the use of implants comprising metal-loaded zeolites for bone grafting, specifically using a combination of citing zinc and copper. In a related approach, US Patent Application Publication 2012/0141429 by Hass describes the use of zinc oxide-coated nanostructures to enhance bone hyperplasia. International Patent Application Publication WO 2011/058443 by Barralet, although not directly referring to implants, describes the administration of various insoluble metal phosphates at sites where enhanced bone mineralization is desired. However, it is unclear whether such methods provide sufficient control over the amount of metal supplied to avoid the release of toxic and/or cytotoxic metal levels.
因此，現仍亟需提出用於以受控方式提供金屬離子源以提高骨密度的組合物及方法。Therefore, there remains a need for compositions and methods for providing a source of metal ions in a controlled manner to increase bone density.
本發明涉及提供組合物及方法，其中係以提供鎂、銅、鍶及鋅離子的混合物以增加骨生長及／或密度。雖然鈣是通常用於支持骨生長及發育的補充物，但是發明人發現，當以一定的莫爾比率施用鎂、銅、鍶及鋅離子時，可以提供增強骨生長及密度的協同效應，而容許使用少量且不造成細胞毒性的各種金屬。鎂、銅、鍶及鋅可以提供作為口服的給藥形式、作為局部製劑、或作為植入物。The present invention is directed to providing compositions and methods wherein a mixture of magnesium, copper, strontium and zinc ions is provided to increase bone growth and/or density. While calcium is a supplement commonly used to support bone growth and development, the inventors have found that magnesium, copper, strontium and zinc ions, when administered in certain molar ratios, can provide a synergistic effect of enhancing bone growth and density, while Various metals are tolerated in small amounts without causing cytotoxicity. Magnesium, copper, strontium, and zinc can be provided as oral administration forms, as topical formulations, or as implants.
在本發明的一實施例中，提出一種用於增強骨生長或密度的礦物質補充劑，該礦物質補充劑包括鎂源、銅源、鋅源、鍶源且被配製成以鎂：鍶：銅：鋅的莫爾比率為0.8-2.4:5.3-15.8:1:1.3-5的方式向受試者提供鎂、鍶、銅及鋅。在一些實施例中，存在於補充劑中的這些金屬的含量可以藉由吸收因子（例如，口服吸收因子、局部吸收因子或植入吸收因子）來調整以提供所需濃度及／或比例的金屬。該礦物質補充劑相較於對應的包括鎂但不具有銅、鋅及鍶的礦物質補充劑，被配製以增加骨密度或骨體積。鎂、鍶、銅及鋅可以作為金屬鹽、金屬複合物及／或含金屬的化合物被提供。在一些實施例中，該礦物質補充劑不包括鈣。在其他實施例中，鎂源是鎂鹽、鎂複合物及／或含鎂化合物。在一些實施例中，鎂源不是有機鎂鹽，銅源可以是銅鹽、銅複合物及／或含銅化合物。在一些實施例中，銅源不是有機銅鹽，鋅源可以是鋅鹽，鋅複合物及／或含鋅化合物。在一些實施例中，鋅源不是有機鋅鹽，鍶源可以是鍶鹽，鍶複合物及／或含鍶化合物。在一些實施例中，鍶源不是有機鍶鹽。In one embodiment of the present invention, there is provided a mineral supplement for enhancing bone growth or density, the mineral supplement comprising a source of magnesium, a source of copper, a source of zinc, a source of strontium and is formulated as magnesium:strontium The subjects were provided with magnesium, strontium, copper, and zinc in such a way that the molar ratio of copper:zinc was 0.8-2.4:5.3-15.8:1:1.3-5. In some embodiments, the levels of these metals present in the supplement can be adjusted by absorption factors (eg, oral absorption factors, topical absorption factors, or implant absorption factors) to provide desired concentrations and/or ratios of the metals . The mineral supplement is formulated to increase bone density or bone volume compared to corresponding mineral supplements including magnesium but without copper, zinc and strontium. Magnesium, strontium, copper and zinc can be provided as metal salts, metal complexes and/or metal containing compounds. In some embodiments, the mineral supplement does not include calcium. In other embodiments, the magnesium source is a magnesium salt, a magnesium complex, and/or a magnesium-containing compound. In some embodiments, the magnesium source is not an organomagnesium salt, and the copper source may be a copper salt, a copper complex, and/or a copper-containing compound. In some embodiments, the copper source is not an organic copper salt, and the zinc source may be a zinc salt, a zinc complex, and/or a zinc-containing compound. In some embodiments, the zinc source is not an organozinc salt, and the strontium source may be a strontium salt, a strontium complex, and/or a strontium-containing compound. In some embodiments, the strontium source is not an organic strontium salt.
在一些實施例中，該礦物質補充劑被配製成可注射的。在其他實施例中，該礦物質補充劑被配製成用於口服施用，且各金屬的含量可藉由口服吸收因子調整為提供吸收鎂：鍶：銅：鋅的莫爾比率為0.8-2.4:5.3-15.8:1:1.3-5。在另一其他實施例中，該礦物質補充劑被配製用於局部施用，且各金屬的含量可藉由局部吸收因子調整為提供吸收鎂：鍶：銅：鋅的莫爾比率為0.8-2.4:5.3-15.8:1:1.3-5。在又一其他實施例中，該礦物質補充劑被配製為植入物（可包括一種可吸收載體），且各金屬的含量可藉由植入吸收因子調整為提供被吸收的鎂：鍶：銅：鋅的莫爾比率為0.8-2.4:5.3-15.8:1:1.3-5。在一些實施例中，該植入物的鎂：鍶：銅：鋅的莫爾比率係為1.2:7.2-7.9:1:2.5。In some embodiments, the mineral supplement is formulated as an injectable. In other embodiments, the mineral supplement is formulated for oral administration and the content of each metal can be adjusted by oral absorption factors to provide absorption at a magnesium:strontium:copper:zinc molar ratio of 0.8-2.4 :5.3-15.8:1:1.3-5. In yet other embodiments, the mineral supplement is formulated for topical application and the content of each metal can be adjusted by topical absorption factors to provide absorption at a magnesium:strontium:copper:zinc molar ratio of 0.8-2.4 :5.3-15.8:1:1.3-5. In yet other embodiments, the mineral supplement is formulated as an implant (which may include an absorbable carrier), and the content of each metal can be adjusted by implant absorption factors to provide absorbed magnesium:strontium: The molar ratio of copper:zinc is 0.8-2.4:5.3-15.8:1:1.3-5. In some embodiments, the implant has a magnesium:strontium:copper:zinc molar ratio of 1.2:7.2-7.9:1:2.5.
本發明的另一實施例，係一種增加骨密度或改善骨生成的方法，其係藉由提供包括鎂源、銅源、鋅源及鍶源的礦物質補充劑及將該礦物質補充劑應用於需要增加骨密度或改善骨生成的個體。該礦物質補充劑被配製成鎂：鍶：銅：鋅的莫爾比率為0.8-2.4:5.3-15.8:1:1.3-5以向該個體提供鎂、銅、鋅及鍶，以及相較於其他包括鎂但不具有銅、鋅及鍶的礦物質補充劑，該礦物質補充劑被配製以增加骨密度或骨體積。在一些實施例中，該礦物質補充劑不包括鈣。在其他實施例中，鎂源不是有機鎂鹽、銅源不是有機銅鹽、鋅源不是有機鋅鹽、且鍶源不是有機鍶鹽。Another embodiment of the present invention is a method of increasing bone density or improving bone formation by providing a mineral supplement including a magnesium source, a copper source, a zinc source and a strontium source and applying the mineral supplement For individuals who need to increase bone density or improve bone formation. The mineral supplement is formulated in a magnesium:strontium:copper:zinc molar ratio of 0.8-2.4:5.3-15.8:1:1.3-5 to provide the individual with magnesium, copper, zinc and strontium, and compared to As with other mineral supplements that include magnesium but not copper, zinc and strontium, the mineral supplement is formulated to increase bone density or bone volume. In some embodiments, the mineral supplement does not include calcium. In other embodiments, the magnesium source is not an organic magnesium salt, the copper source is not an organic copper salt, the zinc source is not an organic zinc salt, and the strontium source is not an organic strontium salt.
礦物質補充劑可以透過注射或輸注包括該礦物質補充劑的溶液來施用。在其他實施例中，該礦物質補充劑透過局部施用包括該礦物質補充劑的洗劑、凝膠、懸浮液或溶液來應用，並且可以藉由對應的局部吸收因子來調整存在的特定金屬的含量以達到所需的鎂：鍶：銅：鋅比率。在另一其他實施例中，該礦物質補充劑透過口服施用包括該礦物質補充劑的丸劑、片劑、膠囊、溶液或懸浮液來應用，並且可以藉由對應的口服吸收因子來調整存在的特定金屬的含量以達到所需的鎂：鍶：銅：鋅比率。在又一其他實施例中，該礦物質補充劑作為手術植入物應用（其中該手術植入物的至少一部分包括該礦物質補充劑），並且可以藉由對應的植入吸收因子來調整存在的特定金屬的含量以達到所需的鎂：鍶：銅：鋅比率。Mineral supplements can be administered by injection or infusion of solutions comprising the mineral supplement. In other embodiments, the mineral supplement is applied by topical application of a lotion, gel, suspension or solution comprising the mineral supplement, and the presence of specific metals can be adjusted by corresponding topical absorption factors level to achieve the desired magnesium:strontium:copper:zinc ratio. In yet other embodiments, the mineral supplement is applied by oral administration of a pill, tablet, capsule, solution or suspension comprising the mineral supplement, and the presence of a corresponding oral absorption factor can be adjusted Amounts of specific metals to achieve the desired magnesium:strontium:copper:zinc ratio. In yet other embodiments, the mineral supplement is applied as a surgical implant (wherein at least a portion of the surgical implant includes the mineral supplement), and the presence can be adjusted by a corresponding implant absorption factor amount of a specific metal to achieve the desired magnesium:strontium:copper:zinc ratio.
根據以下對各實施例的敘述以及圖式內容，將使本發明中的各種目的，特徵，實施態樣和優點更臻顯著，其中相似的元件符號表示相似的元件。Various objects, features, implementations and advantages of the present invention will be more apparent from the following description of the various embodiments and the drawings, wherein like reference numerals refer to like elements.
以下討論提供了本發明的許多示範性實施例，雖然每個實施例都代表本發明元件的單一種組合，仍應認為但本發明仍應包括揭露之元件的所有可能的組合。因此，如果一個實施例包括元件A、B及C，而第二實施例包括元件B及D，即使未明確揭露，本發明仍應理解為包括A、B、C或D的其他剩餘組合。The following discussion provides many exemplary embodiments of the invention, and while each embodiment represents a single combination of elements of the invention, the invention should still be considered to include all possible combinations of the disclosed elements. Thus, if one embodiment includes elements A, B, and C, and a second embodiment includes elements B and D, the invention should be understood to include other remaining combinations of A, B, C, or D even if not explicitly disclosed.
鈣質補充物（通常以口服施用的碳酸鈣形式）長期以來被用於治療或預防骨生長障礙，例如更年期及更年期後的骨質疏鬆症。令人驚奇的是，發明人發現其他金屬（例如鎂及其它金屬鹽）對骨生長及密度具有顯著及意想不到的正面影響，特別是當組合施用時。雖然在沒有鈣補充的情況下就會出現這種效應，但是前述金屬或金屬鹽的組合也可以在有補充鈣及／或其他元素（作為混合金屬製劑的一部分或作為獨立製劑施用）的情況下被提供。Calcium supplements, usually in the form of orally administered calcium carbonate, have long been used to treat or prevent bone growth disorders, such as menopause and postmenopausal osteoporosis. Surprisingly, the inventors discovered that other metals, such as magnesium and other metal salts, have significant and unexpected positive effects on bone growth and density, especially when administered in combination. Although this effect occurs in the absence of calcium supplementation, combinations of the aforementioned metals or metal salts may also be present in the presence of supplemental calcium and/or other elements (either as part of a mixed metal formulation or administered as a separate formulation) Provided.
本揭露提供一種組合物及方法，其係將一非鈣金屬離子（例如鎂、銅、鍶及鋅離子）混合物以一定量及莫爾比率的方式提供，而相較於未處理的組織，具有該含量及莫耳比率的非鈣金屬離子混合物可增加骨生長及／或密度。雖然鈣通常用作支持骨生長及發育的補充劑，但是發明人發現，當以特定的莫耳比率範圍施用時，鎂、銅、鍶及鋅離子可以提供協同效應（即大於加成效應）以增強骨生長及密度。如此獲得的協同效應有利地允許個別金屬以無毒及／或非細胞毒性濃度的方式被使用，同時增強骨生長及／或密度。包括以此處所述的莫耳比率的鎂、銅、鍶及鋅的配方可以在有補充或未補充鈣（例如，鈣的碳酸鹽、碳酸氫鹽及／或氯化物）及／或其它元素（例如鋇、硼、鉀及／或釩）的情況下被提供。這些補充的鈣或其他元素可以成為含鎂、銅、鍶及鋅的配方的一部分，或者分開施用。本揭露提出的配方可以用作輸注、口服給藥形式、作為局部製劑、及／或作為植入物或人造移植物而提供。The present disclosure provides a composition and method for providing a mixture of non-calcium metal ions (eg, magnesium, copper, strontium, and zinc ions) in a certain amount and molar ratio that has, compared to untreated tissue, a This content and molar ratio of the mixture of non-calcium metal ions increases bone growth and/or density. While calcium is commonly used as a supplement to support bone growth and development, the inventors have discovered that magnesium, copper, strontium and zinc ions can provide a synergistic (ie greater than additive effect) effect when administered in a specific molar ratio range to Enhances bone growth and density. The synergistic effect thus obtained advantageously allows the individual metals to be used in non-toxic and/or non-cytotoxic concentrations while enhancing bone growth and/or density. Formulations including magnesium, copper, strontium, and zinc in the molar ratios described herein may be available with or without supplemental calcium (eg, calcium carbonate, bicarbonate, and/or chloride) and/or other elements (eg barium, boron, potassium and/or vanadium). These supplements of calcium or other elements can be part of a formula containing magnesium, copper, strontium, and zinc, or administered separately. The formulations presented in this disclosure can be provided as infusion, oral administration form, as topical formulation, and/or as an implant or artificial graft.
在本揭露所提出的組合物及方法中，係使用四種金屬元素（例如鎂（Mg）、鍶（Sr）、銅（Cu）及鋅（Zn））的組合來誘導骨生成及／或增加骨密度。這些元素可以以離子化合物（即鹽），複合物及／或共價化合物的形式被提供。在一些實施例中，使用局部傳遞（例如局部施用，植入物的使用等）而不是全身傳遞（例如口服施用，靜脈施用等）來提供這些金屬元素。儘管已有許多礦物質組合物被用於相似的目的，但是發明人發現，當以特定的莫爾比率範圍內提供這些金屬元素時，會對骨生成及／或骨密度產生協同作用。In the proposed compositions and methods of the present disclosure, a combination of four metal elements, such as magnesium (Mg), strontium (Sr), copper (Cu), and zinc (Zn), is used to induce and/or increase osteogenesis bone density. These elements can be provided in the form of ionic compounds (ie salts), complexes and/or covalent compounds. In some embodiments, these metal elements are provided using local delivery (eg, topical administration, use of implants, etc.) rather than systemic delivery (eg, oral administration, intravenous administration, etc.). Although many mineral compositions have been used for similar purposes, the inventors have discovered that when these metal elements are provided in a specific molar ratio range, there is a synergistic effect on osteogenesis and/or bone density.
應當理解的是，多種金屬在骨生長修飾中的特定比例的協同作用，有利地允許這些金屬在其濃度遠低於會造成細胞毒性的濃度下的有效地被使用，從而降低了患者的風險，同時保留治療效果。It will be appreciated that the synergistic effect of specific ratios of metals in bone growth modification advantageously allows the effective use of these metals at concentrations well below those that would cause cytotoxicity, thereby reducing the risk to the patient, while preserving the therapeutic effect.
雖然本揭露中所使用的鎂、鍶、銅及鋅的特定鹽類如下所述，但應當理解的是亦可使用該些金屬的任何適合的無機鹽類。舉例來說，前述鎂、鍶、銅及／或鋅鹽的適合的陰離子包括氯離子、氟離子、碳酸根離子、碳酸氫根離子、硫酸根離子、磷酸根離子、硼酸根離子及／或硝酸根離子。在一些實施例中，鎂、鍶、銅及／或鋅可以用氧化物的形式來提供。應當理解的是，可以選擇以金屬鹽及／或氧化物的形式來提供不同比例的金屬。例如，可以在需要長期效果的情況下（例如植入物）選擇相對不溶的鎂、鍶、銅及鋅鹽（例如磷酸鹽及／或硫酸鹽）。或者，可以在需要立即的效果的情況下（例如輸注）選擇相對可溶的鎂、鍶、銅及鋅鹽（例如硝酸鹽及／或氯化物）。While the specific salts of magnesium, strontium, copper, and zinc used in this disclosure are described below, it should be understood that any suitable inorganic salts of these metals may also be used. For example, suitable anions of the aforementioned magnesium, strontium, copper and/or zinc salts include chloride, fluoride, carbonate, bicarbonate, sulfate, phosphate, borate and/or nitric acid root ion. In some embodiments, magnesium, strontium, copper, and/or zinc may be provided as oxides. It will be appreciated that the metals may be chosen to provide different ratios of metals in the form of metal salts and/or oxides. For example, relatively insoluble magnesium, strontium, copper, and zinc salts (eg, phosphates and/or sulfates) may be selected where long-term effects are desired (eg, implants). Alternatively, relatively soluble magnesium, strontium, copper, and zinc salts (eg, nitrates and/or chlorides) may be selected where immediate effects (eg, infusions) are desired.
在一些實施例中，用於描述及要求保護本發明某些實施例之表示成分的量、特性（例如濃度、反應條件等等的數字，應理解為在一些情況下以用語「大約」加以修飾。因此，在一些實施例中，本說明書及所附之申請專利範圍中所記載的數值參數是近似值，其可依據特定實施例所試圖獲得之所需特性而改變。在一些實施例中，應根據所記載之有效位數的數字及應用一般的數值簡化技術來解釋數值參數。儘管闡述本發明一些實施例的廣泛範圍的數值範圍及數值參數是近似值，但在具體實例中所提出的數值則是盡可能地精確記載。在本發明一些實施例中所記載的數值可能包含某些誤差，其係由存在於它們各自試驗測量中的標準差所必然產生的誤差。In some embodiments, numbers representing amounts, properties (eg, concentrations, reaction conditions, etc.) of ingredients used to describe and claim certain embodiments of the invention should be understood to be modified in some cases by the term "about" Accordingly, in some embodiments, the numerical parameters set forth in this specification and the appended claims are approximations that may vary depending upon the desired properties sought to be obtained in a particular embodiment. Numerical parameters are explained according to the number of recorded significant digits and the application of general numerical simplification techniques.Although numerical ranges and numerical parameters of the broad scope of some embodiments of the present invention are set forth as approximations, the numerical values proposed in specific examples are then are reported as precisely as possible.The numerical values set forth in some embodiments of the invention may contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
如在本文所使用的描述及整個要求保護的內容中，除非上下文另有明確規定，否則「一」、「其」及「該」包括複數的含義。此外，如本說明書中所使用的描述，除非上下文另有明確規定，「在」的含義包括「在…內」及「在…上」。As used in the description and throughout the claimed content, "a," "the," and "the" include plural referents unless the context clearly dictates otherwise. Further, as used in the description in this specification, unless the context clearly dictates otherwise, the meaning of "in" includes "within" and "on".
除非上下文另有規定，本文所提出的所有範圍應解釋為包含它們的端點，而開放式範圍應解釋為包含商業上可實施的數值。同樣地，除非上下文另有規定，所有的數值列表應被認為包含其中間的數值。Unless context dictates otherwise, all ranges set forth herein should be construed to include their endpoints, and open-ended ranges should be construed to include commercially feasible values. Likewise, unless context dictates otherwise, all numerical listings should be considered to include intervening numerical values.
本發明中數值範圍的敘述僅意在作為一一指稱各個落在該範圍內的單獨數值的簡寫方法。除非另有說明，在範圍內的每個單獨數值皆被納為本說明書揭露之一部分，如同其於本文中一一被指稱。除非另有說明或者與上下文明顯矛盾，本發明所述的所有方法可以以任何適合的順序進行。在本發明某些實施例中使用的任何和所有範例或示範性語詞（例如「舉例來說」）僅旨在更清楚的說明本發明，並非限制本發明所要求的保護範圍。說明書中的任何語詞不應解釋為未見於申請專利範圍中但為實施本發明的必要元素。The recitation of ranges of values herein are merely intended as a shorthand method of referring to each individual numerical value that falls within the range. Unless otherwise indicated, each individual value within a range is included as part of the disclosure of this specification as if it were individually referred to herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. Any and all exemplary or exemplary words (eg, "for example") used in certain embodiments of the invention are intended only to more clearly describe the invention and do not limit the scope of the invention as claimed. No language in the specification should be construed as an element not found in the claimed claim but as an essential element for the practice of the invention.
本文所揭露之替代元件或實施例之群組不應解釋為本發明之限制。每個群組成員可被單獨地提及和要求保護，或者係以與該群組內其他成員或本文中其他元件任意結合的方式被提及和要求保護。基於便利性及／或專利性的理由，群組中之一個或多個成員可以納入在群組中或自群組中刪除。 當發生任何前述之納入或刪除的情況時，說明書應被視為包括修改後之群組，從而滿足所附之申請專利範圍中使用的所有馬庫西式群組之書面說明要件。Alternate elements or groups of embodiments disclosed herein should not be construed as limitations of the invention. Each group member may be referred to and claimed individually or in any combination with other members of the group or other elements herein. One or more members of the group may be included in or deleted from the group for reasons of convenience and/or patentability. In the event of any of the foregoing inclusions or deletions, the specification shall be deemed to include the groups as amended so as to satisfy the written description requirements of all Markussi-style groups used in the appended claims.
發明人發現細胞毒性作用可以被鎂、鍶、銅及／或鋅離子誘發。四甲基偶氮唑鹽（3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide，MTT）比色法提供了一種可直觀且可量化的方法來確定各種細胞類型的細胞存活率。使用MTT比色法測定人類間質幹細胞中鎂、鍶、銅及／或鋅離子的細胞毒性。在一般的測試中，將2.8×104 個細胞/平方公分（cm2 ）的人類間質幹細胞（hMSC）培養於96孔組織培養盤中，培養液為低葡萄糖DMEM培養液，添加有10%（v/v）胎牛血清（FBS，Biowest，法國）、抗生素（100U/ml青黴素（penicillin）及100 µg/ml鏈黴素（streptomycin））及2 mM的L-麩醯胺酸（L-glutamine）。在細胞培養一天後，將含有不同潛在離子濃度範圍（如表1所示）的測試溶液加入各孔中。在磷酸鹽緩衝溶液（phosphate buffered saline，PBS，OXOID Limited，英國）中加入噻唑蘭（thiazolyl blue tetrazolium bromide）粉末以製備MTT溶液，並在隔天加入10 μL的5 mg/mL MTT溶液。在培養4小時後，於每個孔中加入100 µL 10%十二烷基硫酸鈉（sodium dodecyl sulfate，SDS，西格瑪（Sigma），美國），並在37℃，5% CO2 及95%空氣的環境中孵育至隔天。最後，透過全波長多功能測讀儀記錄570nm波長及640nm參考波長的吸光值，從吸光值量化細胞存活率。The inventors found that cytotoxic effects can be induced by magnesium, strontium, copper and/or zinc ions. The tetramethylazolium salt (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, MTT) colorimetric method provides an intuitive and quantifiable method to determine the cell viability. Cytotoxicity of magnesium, strontium, copper and/or zinc ions in human mesenchymal stem cells using the MTT colorimetric assay. In a typical test, human mesenchymal stem cells (hMSCs) at 2.8×10 4 cells/cm 2 were cultured in 96-well tissue culture dishes in low-glucose DMEM supplemented with 10% (v/v) fetal bovine serum (FBS, Biowest, France), antibiotics (100 U/ml penicillin and 100 µg/ml streptomycin) and 2 mM L-glutamic acid (L- glutamine). After one day of cell culture, test solutions containing different potential ion concentration ranges (as shown in Table 1) were added to each well. MTT solution was prepared by adding thiazolyl blue tetrazolium bromide powder to phosphate buffered saline (PBS, OXOID Limited, UK) and 10 μL of 5 mg/mL MTT solution on alternate days. After 4 hours of incubation, 100 µL of 10% sodium dodecyl sulfate (sodium dodecyl sulfate, SDS, Sigma, USA) was added to each well and incubated at 37°C, 5% CO 2 and 95% air Incubate in the environment until the next day. Finally, the absorbance values of 570nm wavelength and 640nm reference wavelength were recorded by a full-wavelength multi-function reader, and the cell viability was quantified from the absorbance values.
表
鎂、鍶、銅及／或鋅的細胞毒性係以離子形式進行評估，並進行獨立測試以分別找出不會產生細胞毒性作用的有效濃度範圍。結果如圖1A至1D所示。圖1A顯示使用鎂離子進行研究的典型結果。圖1B顯示使用銅離子進行研究的典型結果。圖1C顯示使用鋅離子進行研究的典型結果。圖1D顯示使用鍶離子進行研究的典型結果。通常，當鎂離子濃度為50至200 ppm時，可以觀察到人類間質幹細胞（hMSC）的最大存活率；當濃度超過400 ppm時，存活率開始下降。當鎂離子濃度超過1000 ppm時，大多數細胞死亡。人類間質幹細胞只能在濃度為5至10 ppm的銅離子中存活；當濃度為25 ppm或更高時，存活率下降。令人驚訝的是，人類間質幹細胞（hMSC）對鋅離子特別敏感，當鋅離子濃度超過1 ppm時，發現明顯的細胞死亡。另一方面，人類間質幹細胞被發現對鍶離子具有耐受性，因為高達1000 ppm鍶+處理的細胞仍有接近100%的存活率。令人驚訝的是，實驗結果顯示在100至400 ppm的鍶離子濃度下，人類間質幹細胞的存活率通常增加到115%（相對於對照組細胞）。The cytotoxicity of magnesium, strontium, copper and/or zinc is assessed in ionic form and independently tested to find effective concentration ranges that do not produce cytotoxic effects. The results are shown in Figures 1A to 1D. Figure 1A shows typical results from studies using magnesium ions. Figure 1B shows typical results from studies using copper ions. Figure 1C shows typical results from studies using zinc ions. Figure ID shows typical results from studies using strontium ions. Typically, the maximum survival rate of human mesenchymal stem cells (hMSCs) is observed when magnesium ion concentrations are between 50 and 200 ppm; when the concentration exceeds 400 ppm, the survival rate begins to decline. When the magnesium ion concentration exceeds 1000 ppm, most cells die. Human mesenchymal stem cells can only survive in concentrations of 5 to 10 ppm of copper ions; when the concentration is 25 ppm or higher, the survival rate decreases. Surprisingly, human mesenchymal stem cells (hMSCs) were particularly sensitive to zinc ions, and significant cell death was found when zinc ion concentrations exceeded 1 ppm. On the other hand, human mesenchymal stem cells were found to be resistant to strontium ions, as cells treated with up to 1000 ppm strontium+ still had nearly 100% viability. Surprisingly, the experimental results showed that the survival rate of human mesenchymal stem cells generally increased to 115% (relative to control cells) at strontium ion concentrations of 100 to 400 ppm.
鎂、鍶、銅及／或鋅對人類間質幹細胞成骨分化的影響，係利用細胞的鹼性磷酸酶（ALP）活性作為分化的指標來進行測定。因此，鎂、鍶、銅及／或鋅的成骨分化特性，無論是個別地或組合地，均可使用ALP測定來進行檢測。在典型的ALP測定中，第一天先將1×104 hMSC細胞/平方公分（cm2 ）培養於含有10%（v/v）胎牛血清（FBS，Biowest，法國）、抗生素（100 U/ml青黴素及100 µg/ml鏈黴素）及2 mM的L-麩醯胺酸的低葡萄糖DMEM培養液中。第二天，用分化DMEM（differentiation DMEM）培養液替換各孔中的培養液，分化DMEM培養液含有50 µg/ml抗壞血酸（西格瑪，美國）、10 mM的β-甘油磷酸（β-glycerol phosphate，MP Biomedicals，法國）及0.1 μM迪皮質醇（dexamethasone，西格瑪，美國）以及不同組合的鎂、鍶、銅及／或鋅離子，並在37℃，5% CO2 的環境下孵育3、7及14天。各離子及試驗組的測試濃度分別顯示於表2及表3。這些培養液每3天更換一次。培養後，用磷酸鹽緩衝溶液（PBS）清洗細胞3次，並用0.1%Triton X-100在4℃下裂解30分鐘。將細胞裂解液在574重力加速度（g）及4℃下離心10分鐘（2-5莎多利斯（Sartorius），西格瑪，美國）。將每個樣品的上清液（10 μl）轉移到96孔組織培養盤中。以比色法來測定ALP活性；其係使用含有對硝基苯基磷酸鹽（p-nitrophenyl phosphate，p-NPP）作為受質的ALP試劑（斯德必優（Stanbio），美國）來進行。透過全波長多功能測讀儀Beckman Coulter DTX 880記錄波長為405nm時的吸光值。將觀察到的ALP活性以樣品的總蛋白質濃度進行標準化，總蛋白質濃度係使用Bio-Rad蛋白測定（Bio-Rad，美國）法進行測定。The effects of magnesium, strontium, copper and/or zinc on the osteogenic differentiation of human mesenchymal stem cells were determined by using the alkaline phosphatase (ALP) activity of the cells as an indicator of differentiation. Thus, the osteogenic differentiation properties of magnesium, strontium, copper and/or zinc, either individually or in combination, can be examined using the ALP assay. In a typical ALP assay, 1 x 10 4 hMSC cells/cm 2 were cultured on the first day in cells containing 10% (v/v) fetal bovine serum (FBS, Biowest, France), antibiotics (100 U). /ml penicillin and 100 µg/ml streptomycin) and 2 mM L-glutamic acid in low glucose DMEM medium. The next day, the medium in each well was replaced with differentiation DMEM (differentiation DMEM) medium containing 50 µg/ml ascorbic acid (Sigma, USA), 10 mM β-glycerol phosphate, MP Biomedicals, France) and 0.1 μM dicortisol (dexamethasone, Sigma, USA) and various combinations of magnesium, strontium, copper and/or zinc ions, and incubated at 37°C, 5% CO for 3 , 7 and 14 days. The test concentrations of each ion and test group are shown in Table 2 and Table 3, respectively. These cultures were changed every 3 days. After incubation, cells were washed 3 times with phosphate buffered saline (PBS) and lysed with 0.1% Triton X-100 for 30 min at 4 °C. Cell lysates were centrifuged (2-5 Sartorius, Sigma, USA) at 574 g for 10 min at 4°C. Transfer the supernatant (10 μl) of each sample to a 96-well tissue culture dish. ALP activity was measured colorimetrically; it was performed using ALP reagent (Stanbio, USA) containing p-nitrophenyl phosphate (p-NPP) as a substrate. Absorbance values at a wavelength of 405 nm were recorded through a Beckman Coulter DTX 880, a full-wavelength multifunction reader. The observed ALP activity was normalized to the total protein concentration of the samples, which was determined using the Bio-Rad Protein Assay (Bio-Rad, USA).
表
表
成骨分化研究的典型結果顯示在圖2A及2B中。圖2A顯示從以不同組合及濃度的銅、鋅、鍶及鎂離子處理的細胞中，所測量到的典型標準化ALP活性。圖2B顯示以鎂離子處理的細胞的典型結果。令人驚訝的是，直至開始處理後的14天，相較於對照組細胞（成骨分化的指標），以銅、鋅、鍶及鎂離子的組合進行處理，細胞的ALP活性仍維持增長。前述結果係以簡單鹽類形式來提供銅、鋅、鍶及鎂，並且培養環境中不具有該些金屬的有機鹽類（例如含有對成骨作用具有藥效的有機化合物的銅、鋅、鍶及鎂有機鹽類）。申請人認為此種分化結果將導致體內骨密度及／或骨含量的增加。而其他以兩種或三種前述離子的組合進行處理的結果，並沒有發現成骨分化的增加。這表示銅、鋅、鍶及鎂的組合是必需的。此外，以前述四種離子的組合來處理並產生有效結果的組別中，個別離子的濃度係遠低於其會產生細胞毒性的濃度。值得注意的是，結果顯示其係不需要補充鈣（Ca）及／或磷酸鹽。結果也顯示，其亦無須複合有機化合物。Typical results of osteogenic differentiation studies are shown in Figures 2A and 2B. Figure 2A shows typical normalized ALP activity measured from cells treated with different combinations and concentrations of copper, zinc, strontium and magnesium ions. Figure 2B shows typical results for cells treated with magnesium ions. Surprisingly, the ALP activity of the cells treated with the combination of copper, zinc, strontium and magnesium ions remained increased compared to the control cells (an indicator of osteogenic differentiation) up to 14 days after the initiation of treatment. The foregoing results provide copper, zinc, strontium, and magnesium in the form of simple salts that do not have organic salts of these metals in the culture environment (eg, copper, zinc, strontium, which contain organic compounds that are effective in osteogenesis). and magnesium organic salts). Applicants believe that such differentiation results in an increase in bone density and/or bone content in vivo. While other treatments with a combination of two or three of the aforementioned ions did not find an increase in osteogenic differentiation. This means that a combination of copper, zinc, strontium and magnesium is required. Furthermore, in groups where treatment with a combination of the aforementioned four ions produced effective results, the concentrations of the individual ions were well below the concentrations at which they would be cytotoxic. Notably, the results showed that it does not require calcium (Ca) and/or phosphate supplementation. The results also show that it also does not require complex organic compounds.
發明人已經確定類似的金屬元素組合在體內局部傳遞／施打時也是有效的。為了要在體內傳遞該等金屬元素的組合，可以將銅、鋅、鍶及鎂的鹽類與具有生物相容性的載體（例如聚己內酯（polycaprolactone，PCL））進行組合。在典型的應用中，所選擇的金屬鹽類可與聚己內酯以特定含量來混合。在典型的研究中，植入物係由前述材料製成直徑為2 mm，長度為6 mm的桿體。初步研究中所使用的植入物的金屬含量如表4所示。The inventors have determined that similar combinations of metal elements are also effective for local delivery/administration in vivo. To deliver these combinations of metal elements in vivo, salts of copper, zinc, strontium and magnesium can be combined with biocompatible carriers such as polycaprolactone (PCL). In typical applications, the selected metal salts may be mixed with polycaprolactone at specified levels. In a typical study, the implants were fabricated from the aforementioned materials with a 2 mm diameter and 6 mm length rod. The metal content of the implants used in the preliminary study is shown in Table 4.
表
在典型的研究中，係將前述植入物植入2個月大的雌性SD大鼠（Sprague-Dawley rats，香港大學實驗動物單位，平均體重為200-250g）體內，選擇的手術部位為外上髁（lateral epicondyle）。每隻大鼠在右側外上髁上植入離子組合(鎂/鍶/銅/鋅)/聚己內酯或鎂/聚己內酯複合物。為了監測植入物周圍的新骨形成，係於1、2、3、4及8週的時間點進行連續監測。鎂/聚己內酯複合物則作為對照組。In a typical study, the aforementioned implants were implanted into 2-month-old female SD rats (Sprague-Dawley rats, Experimental Animal Unit, University of Hong Kong, with an average body weight of 200-250 g), and the selected surgical site was external Epicondyle (lateral epicondyle). Each rat was implanted with an ionic combination (magnesium/strontium/copper/zinc)/polycaprolactone or magnesium/polycaprolactone complex on the right lateral epicondyle. To monitor the formation of new bone around the implant, continuous monitoring was performed at time points of 1, 2, 3, 4 and 8 weeks. The magnesium/polycaprolactone complex served as a control.
大鼠以腹膜內注射***（ketamine，67mg/kg）及賽拉嗪（xylazine，6mg/kg）進行麻醉。大鼠的手術部位係經過剃毛並去皮。以微創方法，使用手鑽在外側上髁處製造直徑為2 mm，深度為6 mm的孔。隨後，將樣品植入以前述方式於大鼠右股骨上所鑽好的孔中。然後將傷口逐層縫合，並在切口上施加適當的敷料。手術後，對大鼠皮下注射1 mg/kg土霉素（terramycin）（抗生素）及0.5 mg/kg酮洛芬（ketoprofen）。大鼠在術後8週安樂死。Rats were anesthetized by intraperitoneal injection of ketamine (ketamine, 67 mg/kg) and xylazine (xylazine, 6 mg/kg). The surgical site of the rat was shaved and peeled. In a minimally invasive manner, a hole with a diameter of 2 mm and a depth of 6 mm was made in the lateral epicondyle using a hand drill. Subsequently, the samples were implanted in the holes drilled in the rat's right femur in the manner previously described. The wound is then sutured layer by layer and an appropriate dressing is applied over the incision. After surgery, rats were injected subcutaneously with 1 mg/kg terramycin (an antibiotic) and 0.5 mg/kg ketoprofen. Rats were euthanized 8 weeks after surgery.
在各個時間點（即1、2、3、4及8週），在手術部位進行微電腦斷層掃描（micro-computed tomography，micro-CT）（SKYSCAN 1076，Skyscan Company）以監測癒合過程並檢測植入物周圍的新骨形成。使用NRecon（Skyscan Company）重建二維平面。At various time points (ie, 1, 2, 3, 4, and 8 weeks), micro-computed tomography (micro-CT) (SKYSCAN 1076, Skyscan Company) was performed at the surgical site to monitor the healing process and detect implantation New bone formation around the object. Two-dimensional planes were reconstructed using NRecon (Skyscan Company).
在一些研究中，為了進一步評估釋放的離子對骨生成的影響，除了監測植入物周圍的新骨形成之外，還測量皮質骨的厚度。以沒有植入的肢體作為對照組。在第4週及第8週進行微電腦斷層掃描以監測新骨形成及皮質骨厚度。在這兩個時間點，以微電腦斷層掃描裝置（SKYSCAN 1076，Skyscan Company）掃描大鼠，並透過CTAn軟體（Skyscan Company）分析骨體積。In some studies, to further assess the effect of released ions on osteogenesis, cortical bone thickness was measured in addition to monitoring new bone formation around the implant. The limbs without implantation were used as the control group. Microcomputed tomography scans were performed at
在其他研究中，所取得的股骨的彎曲硬度及強度性質係以基於ASTM D7264-01標準方案的3點彎曲試驗（MTS 858.02Mini Bionix）來測定。測試速度為1 mm/min。In other studies, the flexural stiffness and strength properties of the obtained femurs were determined by the 3-point bending test (MTS 858.02 Mini Bionix) based on the ASTM D7264-01 standard protocol. The test speed is 1 mm/min.
用單一種濃度組合（如表4所述）的鎂、鍶、銅及鋅來進行初步研究的典型結果顯示於圖4A及4B。圖4A顯示從植入樣品的大鼠由第0天到術後第8週其股骨的典型micro-CT影像。圖4B顯示與樣品植入物相鄰的骨體積的變化百分比。通常在術後第2、4、8週時觀察到與鎂/鍶/銅/鋅植入物相鄰（相對於僅含鎂的對照組）的骨體積增加了40%-87%。這表示局部施用的鎂、鍶、銅及鋅組合在誘發新骨形成方面非常有效。值得注意的是，前述結果是在沒有提供鈣或磷酸鹽補充物的情形下發生。此外，以簡單的化學鹽（對比於與具有藥學活性的有機化合物所形成的金屬鹽）形式來補充銅、鋅、鎂及鍶時，仍能產生有效的效果。Typical results from preliminary studies conducted with a single concentration combination (described in Table 4) of magnesium, strontium, copper and zinc are shown in Figures 4A and 4B. Figure 4A shows a typical micro-CT image of the femur from
以類似的研究方式來確定金屬離子的最大劑量。樣品製備、外科過程及手術後Micro-CT掃描與圖4A及4B中概述的研究大致相同。所使用的植入物其金屬鹽含量顯示於表5A及5B中，其係分別顯示金屬元素的重量及比例。The maximum dose of metal ions was determined in a similar manner. Sample preparation, surgical procedures, and post-operative Micro-CT scans were largely the same as the studies outlined in Figures 4A and 4B. The metal salt contents of the implants used are shown in Tables 5A and 5B, which show the weight and proportion of the metal elements, respectively.
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5B5B
此處所用的第一及第二組（即鎂及鎂/鍶/銅/鋅）中各金屬的含量與前述研究中使用的相同，而第三組（即 (鎂/鍶/銅/鋅)+）及第四組（即 (鎂/鍶/銅/鋅)++）中則提高了某些金屬離子（如表5所示）的濃度，提高的濃度範圍為2倍至10倍。以不含金屬離子的聚己內酯作為對照組。通常，鎂/鍶/銅/鋅組在整個植入期間，分別相較於鎂/聚己內酯及僅含聚己內酯的植入物，骨含量增加至少30%至50%（參見圖5）。然而，當使用(鎂/鍶/銅/鋅)+及(鎂/鍶/銅/鋅)++植入物時，在手術後一周發現骨流失25%或以上。雖然骨流失在之後的時間點有所減少，但是於植入前述植入物的組別的實驗結果中也沒有發現新骨形成。此一結果表示新骨形成會被較高濃度的金屬離子所抑制。The first and second groups (i.e. magnesium and magnesium/strontium/copper/zinc) used here have the same metal content as those used in the previous study, while the third group (i.e. (magnesium/strontium/copper/zinc) +) and the fourth group (ie (magnesium/strontium/copper/zinc)++) increased the concentration of some metal ions (as shown in Table 5), the increased concentration ranged from 2-fold to 10-fold. Polycaprolactone without metal ions was used as the control group. In general, the magnesium/strontium/copper/zinc group had at least a 30% to 50% increase in bone content over the entire implant period compared to the magnesium/polycaprolactone and polycaprolactone-only implants, respectively (see Fig. 5). However, when using (Mg/Strontium/Cu/Zn)+ and (Mg/Strontium/Cu/Zn)++ implants, bone loss of 25% or more was found one week after surgery. Although bone loss was reduced at later time points, no new bone formation was found in the experimental results of the groups implanted with the aforementioned implants. This result indicates that new bone formation is inhibited by higher concentrations of metal ions.
以類似的研究方法來進一步優化植入物的金屬鹽含量的濃度。在這些研究中使用的植入物的金屬鹽組成顯示於表6A及6B中。表6A顯示植入物中的鹽重量，而表6B顯示植入物中金屬元素彼此之間的比例。在本研究中，植入物的直徑為1.3 mm，長度為2 cm，形狀呈桿狀。此外，植入部位位於股骨髓內而不是外上髁，以測試整個股骨的機械性質。Similar research methods were used to further optimize the concentration of the metal salt content of the implants. The metal salt compositions of the implants used in these studies are shown in Tables 6A and 6B. Table 6A shows the salt weights in the implants, while Table 6B shows the ratios of the metal elements in the implants to each other. In this study, the implants were 1.3 mm in diameter, 2 cm in length, and rod-like in shape. In addition, the implantation site was located within the femoral bone marrow rather than the lateral epicondyle to test the mechanical properties of the entire femur.
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6B6B
外科手術操作如後。將遠端股骨露出直到看到髕骨，然後將髕骨肌腱移動到外上髁，穿過外上髁在右股骨的遠端鑽出直徑為0.5 mm，長度為3 mm的孔，然後將樣品植入到孔中。左側（無植入）作為對照組。然後，將傷口逐層縫合，並在切口上施加適當的敷料。手術後，對大鼠皮下注射1 mg/kg土霉素（抗生素）及0.5 mg/kg酮洛芬。大鼠在術後8週安樂死。The surgical procedure is as follows. The distal femur is exposed until the patella is visible, then the patellar tendon is moved to the lateral epicondyle, a hole 0.5 mm in diameter and 3 mm in length is drilled through the lateral epicondyle in the distal end of the right femur, and the sample is implanted into the hole. The left side (without implantation) served as the control group. The wound is then sutured layer by layer and an appropriate dressing is applied over the incision. After surgery, rats were injected subcutaneously with 1 mg/kg oxytetracycline (an antibiotic) and 0.5 mg/kg ketoprofen. Rats were euthanized 8 weeks after surgery.
由於鎂及鍶離子在體內大量存在，因此與銅及鋅離子相比，這些離子的耐受性可被認為是相對較高的。發明人考慮到銅及鋅的濃度可能是至關重要的，因此，進一步的優化研究專注在銅及／或鋅的優化。將用各種含量的鎂、鍶、銅及鋅配製的植入物（310）以前述之手術方式植入股骨的髓內管（intramedullary canal）中（如圖3所示），以研究組合金屬離子對皮質骨厚度的影響。以無植入的股骨作為對照組。典型結果如圖6A、6B及6C所示。圖6A顯示皮質骨體積的變化百分比，並且顯示所測試的所有鎂、鍶、銅及鋅製劑相對於無植入及僅含鎂的對照組而言，其平均皮質骨體積增加。令人驚訝的是，相較於對照組，所有經測試的鎂、鍶、銅及鋅組合，其平均骨體積皆增加。如表6B所示，鎂、鍶及鋅的莫耳比率相對於銅含量的範圍分別可為0.8至2.4、5.3至15.8、1.2至5，並可提供前述的效果。特別地，相較於鎂/聚己內酯對照組，在植入植入物製劑MSCZ 2、MSCZ 3及MSCZ 8的組別中，結果顯示其有顯著更高的皮質骨體積，術後第4週係增加4-8%，術後第8週則增加6-11%。此外，植入物製劑MSCZ 7在植入8週後相較於鎂/聚己內酯對照組也顯示顯著較高的皮質骨厚度，約為9%。圖6C及圖6C分別顯示了植入及無植入股骨的彈性係數及硬度。與對照組相比，在植入MSCZ 3植入物的組別中，結果顯示出顯著更高的彈性係數及硬度。通常，相較於股骨沒有手術植入的情況下，在植入MSCZ 3植入物的股骨中，結果顯示彈性係數增加16%，硬度則增加14%。因此，MSCZ 2（鎂：鍶：銅：鋅 = 2.4:15.8:1:5）、MSCZ 3（鎂：鍶：銅：鋅 = 1.0:6.3:1:2）、MSCZ 7（鎂：鍶：銅：鋅 = 1.2:7.9:1:3.1）及MSCZ 8（鎂：鍶：銅：鋅 = 1.2:7.9:1:3.8）植入物對骨生成特別有效。Since magnesium and strontium ions are abundant in the body, the tolerance of these ions can be considered to be relatively high compared to copper and zinc ions. The inventors considered that the concentration of copper and zinc may be critical, therefore, further optimization studies focused on the optimization of copper and/or zinc. Implants (310) formulated with various levels of magnesium, strontium, copper, and zinc were surgically implanted into the intramedullary canal of the femur (shown in Figure 3) as previously described to study the combined metal ions Effects on cortical bone thickness. The non-implanted femur was used as the control group. Typical results are shown in Figures 6A, 6B and 6C. Figure 6A shows the percent change in cortical bone volume and shows an increase in mean cortical bone volume for all magnesium, strontium, copper, and zinc formulations tested relative to the non-implanted and magnesium-only controls. Surprisingly, mean bone volume was increased for all magnesium, strontium, copper and zinc combinations tested compared to the control group. As shown in Table 6B, the molar ratios of magnesium, strontium, and zinc can range from 0.8 to 2.4, 5.3 to 15.8, and 1.2 to 5, respectively, with respect to the copper content, and can provide the aforementioned effects. In particular, in the groups implanted with
雖然上述的實施例是利用植入物，但是本揭露提出的其它實施例包括提供鎂、鍶、銅及／或鋅的局部傳遞而非使用植入物的方法、裝置及組合物。在該實施例中，鎂、鍶、銅及／或鋅可以以可流動的劑型來提供，例如凝膠或其他半固體，其可以在需要增加或增強骨生成的部位或附近施用。舉例來說，這種凝膠或半固體可以透過注射或微創外科手術引入。在一些實施例中，可流動製劑可以在施用後固化，以增強定位。在較佳地實施例中，該可流動製劑是可被個體吸收的。While the above-described embodiments utilize implants, other embodiments proposed by the present disclosure include methods, devices, and compositions that provide localized delivery of magnesium, strontium, copper, and/or zinc rather than using implants. In this embodiment, the magnesium, strontium, copper and/or zinc can be provided in a flowable dosage form, such as a gel or other semisolid, which can be administered at or near the site where increased or enhanced osteogenesis is desired. For example, such gels or semisolids can be introduced by injection or minimally invasive surgery. In some embodiments, the flowable formulation can be cured after application to enhance positioning. In preferred embodiments, the flowable formulation is ingestible by the subject.
在本揭露的其它實施例中，可以全身性地提供鎂、鍶、銅及／或鋅。適合的全身性製劑的實例包括適用於腸胃外施用的注射劑及可以口服的製劑。適合的口服製劑可以是液體、固體或半固體。在該口服製劑中，提供的鎂、鍶、銅及／或鋅的量及／或比例可以利用吸收因子來調整至所需含量及／或這些離子的莫耳比率。例如，應用於口服的水溶性鎂鹽的吸收因子約是30%。類似地，應用於口服的鍶的吸收因子可為約15%至約30%。應用於口服的鋅的吸收因子可為約20%至40%。在一些實施例中，吸收因子可以隨著所提供的鎂、鍶、銅及／或鋅的含量而變化。例如，當銅含量降低時，口服攝取的銅的吸收因子可以在約30%至約65%的範圍內。在一些實施例中，吸收可以根據製劑中使用的特定鎂、鍶、銅及／或鋅化合物而變化。對於局部施用製劑及植入物型態的製劑，可以認為有近似的吸收因子，並將該吸收因子應用於這些製劑以提供所需的銅：鋅：鍶：鎂的莫耳比率。In other embodiments of the present disclosure, magnesium, strontium, copper, and/or zinc may be provided systemically. Examples of suitable systemic formulations include injections suitable for parenteral administration and formulations that can be taken orally. Suitable oral formulations may be liquid, solid or semi-solid. In the oral formulation, the amount and/or ratio of magnesium, strontium, copper and/or zinc provided can be adjusted to the desired content and/or molar ratio of these ions using absorption factors. For example, the absorption factor of water-soluble magnesium salts for oral application is about 30%. Similarly, the absorption factor of strontium applied orally may be from about 15% to about 30%. The absorption factor of zinc applied orally may be about 20% to 40%. In some embodiments, the absorption factor may vary with the amount of magnesium, strontium, copper, and/or zinc provided. For example, the absorption factor of orally ingested copper may range from about 30% to about 65% when the copper content is reduced. In some embodiments, absorption can vary depending on the specific magnesium, strontium, copper and/or zinc compounds used in the formulation. For both topical formulations and implant-type formulations, an approximate absorption factor can be considered and applied to these formulations to provide the desired copper:zinc:strontium:magnesium molar ratio.
在本揭露的一些實施例中，鎂、鍶、銅及／或鋅可以與可以支持骨形成及／或抑制骨吸收的額外元素一起提供。這些額外元素包括鋇、硼、鈣，鉀及／或釩。這些額外元素可以以有機或無機鹽、複合物或化合物的形式來提供。例如，鋇、硼、鈣，鉀及／或釩可以以鹽類中的陽離子形式被提供。前述的鹽類可以包括如氯離子、氟離子、碳酸根離子、碳酸氫根離子、硫酸根離子、磷酸根離子、硼酸根離子或硝酸根離子等陰離子。在另一個實施例中，硼可以以硼酸鹽及／或硼／醣（碳水化合物）複合物的形式來提供。In some embodiments of the present disclosure, magnesium, strontium, copper, and/or zinc may be provided with additional elements that may support bone formation and/or inhibit bone resorption. These additional elements include barium, boron, calcium, potassium and/or vanadium. These additional elements may be provided in the form of organic or inorganic salts, complexes or compounds. For example, barium, boron, calcium, potassium and/or vanadium can be provided as cations in salts. The aforementioned salts may include anions such as chloride, fluoride, carbonate, bicarbonate, sulfate, phosphate, borate or nitrate. In another embodiment, boron can be provided in the form of borate salts and/or boron/sugar (carbohydrate) complexes.
本發明所屬技術領域具有通常知識者應當明瞭，除了前述已記載的實施例之外，可能存在其他不偏離本發明概念的修改。因此，任何未脫離本發明之精神與範疇，而對其進行之等效修改或變更，均應包含於後附之申請專利範圍中。此外，在解釋說明書及申請專利範圍兩者時，所有技術用語應以與上下文一致之最廣的可能方式解釋。特別是用語「包含」及「包括」，應解釋為其係以非排他性的形式指稱元件、成分或步驟，其係表示所指稱之元件、成分或步驟可與其他未被明確提及之元件、成分或步驟一起存在、使用或結合。在說明書或申請專利範圍中提及某物的至少其中之一係選自由A、B、C…及N所組成之群組時，該內容應解釋為存在該群組其中的一個元件即可，而非必須要有A加N、或B加N等等。It should be apparent to those skilled in the art to which the present invention pertains that, in addition to the previously described embodiments, there may be other modifications that do not depart from the concept of the present invention. Therefore, any equivalent modifications or changes without departing from the spirit and scope of the present invention should be included in the appended patent application scope. Furthermore, in interpreting both the specification and the claimed scope, all technical terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprising" and "comprising" should be construed as referring to an element, component or step in a non-exclusive manner, which means that the referred element, component or step may be combined with other elements, components or steps not explicitly mentioned, The ingredients or steps are present, used or combined together. When it is mentioned in the description or the scope of the patent application that at least one of something is selected from the group consisting of A, B, C... and N, the content should be interpreted as the existence of one element in the group, It is not necessary to have A plus N, or B plus N, etc.
參考文獻 [1] Tao ZS, Zhou WS, He XW, Liu W, Bai BL, Zhou Q, et al. A comparative study of zinc, magnesium, strontium-incorporated hydroxyapatite-coated titanium implants for osseointegration of osteopenic rats. Materials Science & Engineering C-Materials for Biological Applications. 2016;62:226-32. [2] He LY, Zhang XM, Liu B, Tian Y, Ma WH. Effect of magnesium ion on human osteoblast activity. Braz J Med Biol Res. 2016;49. [3] Yoshizawa S, Brown A, Barchowsky A, Sfeir C. Magnesium ion stimulation of bone marrow stromal cells enhances osteogenic activity, simulating the effect of magnesium alloy degradation. Acta Biomater. 2014;10:2834-42. [4] Castellani C, Lindtner RA, Hausbrandt P, Tschegg E, Stanzl-Tschegg SE, Zanoni G, et al. Bone-implant interface strength and osseointegration: Biodegradable magnesium alloy versus standard titanium control. Acta Biomaterialia. 2011;7:432-40. [5] Wang GF, Li JH, Zhang WJ, Xu LY, Pan HY, Wen J, et al. Magnesium ion implantation on a micro/nanostructured titanium surface promotes its bioactivity and osteogenic differentiation function. International Journal of Nanomedicine. 2014;9:2387-98. [6] Meininger S, Mandal S, Kumar A, Groll J, Basu B, Gbureck U. Strength reliability and in vitro degradation of three-dimensional powder printed strontium-substituted magnesium phosphate scaffolds. Acta Biomaterialia. 2016;31:401-11. [7] Gentleman E, Fredholm YC, Jell G, Lotfibakhshaiesh N, O'Donnell MD, Hill RG, et al. The effects of strontium-substituted bioactive glasses on osteoblasts and osteoclasts in vitro. Biomaterials. 2010;31:3949-56. [8] Zhang W, Shen Y, Pan H, Lin K, Liu X, Darvell BW, et al. Effects of strontium in modified biomaterials. Acta Biomater. 2011;7:800-8. [9] Boda SK, Thrivikraman G, Panigrahy B, Sarma DD, Basu B. Competing Roles of Substrate Composition, Microstructure, and Sustained Strontium Release in Directing Osteogenic Differentiation of hMSCs. ACS Appl Mater Interfaces. 2016. [10] Yusa K, Yamamoto O, Takano H, Fukuda M, Iino M. Zinc-modified titanium surface enhances osteoblast differentiation of dental pulp stem cells in vitro. Sci Rep-Uk. 2016;6. [11] Ito A, Otsuka M, Kawamura H, Ikeuchi M, Ohgushi H, Sogo Y, et al. Zinc-containing tricalcium phosphate and related materials for promoting bone formation. Current Applied Physics. 2005;5:402-6. [12] Wang T, Zhang JC, Chen Y, Xiao PG, Yang MS. Effect of zinc ion on the osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells and the adipocytic trans-differentiation of mouse primary osteoblasts. Journal of Trace Elements in Medicine and Biology. 2007;21:84-91. [13] Seo HJ, Cho YE, Kim T, Shin HI, Kwun IS. Zinc may increase bone formation through stimulating cell proliferation, alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells. Nutr Res Pract. 2010;4:356-61. [14] Jin SJ, Ren L, Yang K. Bio-Functional Cu Containing Biomaterials: a New Way to Enhance Bio-Adaption of Biomaterials. Journal of Materials Science & Technology. 2016;32:835-9. [15] Wu CT, Zhou YH, Xu MC, Han PP, Chen L, Chang J, et al. Copper-containing mesoporous bioactive glass scaffolds with multifunctional properties of angiogenesis capacity, osteostimulation and antibacterial activity. Biomaterials. 2013;34:422-33. [16] Sen CK, Khanna S, Venojarvi M, Trikha P, Ellison EC, Hunt TK, et al. Copper-induced vascular endothelial growth factor expression and wound healing. Am J Physiol-Heart C. 2002;282:H1821-H7. [17] Shi MC, Chen ZT, Farnaghi S, Friis T, Mao XL, Xiao Y, et al. Copper-doped mesoporous silica nanospheres, a promising immunomodulatory agent for inducing osteogenesis. Acta Biomaterialia. 2016;30:334-44.references [1] Tao ZS, Zhou WS, He XW, Liu W, Bai BL, Zhou Q, et al. A comparative study of zinc, magnesium, strontium-incorporated hydroxyapatite-coated titanium implants for osseointegration of osteopenic rats. Materials Science & Engineering C-Materials for Biological Applications. 2016;62:226-32. [2] He LY, Zhang XM, Liu B, Tian Y, Ma WH. Effect of magnesium ion on human osteoblast activity. Braz J Med Biol Res. 2016;49. [3] Yoshizawa S, Brown A, Barchowsky A, Sfeir C. Magnesium ion stimulation of bone marrow stromal cells enhances osteogenic activity, simulating the effect of magnesium alloy degradation. Acta Biomater. 2014;10:2834-42. [4] Castellani C, Lindtner RA, Hausbrandt P, Tschegg E, Stanzl-Tschegg SE, Zanoni G, et al. Bone-implant interface strength and osseointegration: Biodegradable magnesium alloy versus standard titanium control. Acta Biomaterialia. 2011;7:432 -40. [5] Wang GF, Li JH, Zhang WJ, Xu LY, Pan HY, Wen J, et al. Magnesium ion implantation on a micro/nanostructured titanium surface promotes its bioactivity and osteogenic differentiation function. International Journal of Nanomedicine. 2014;9 :2387-98. [6] Meininger S, Mandal S, Kumar A, Groll J, Basu B, Gbureck U. Strength reliability and in vitro degradation of three-dimensional powder printed strontium-substituted magnesium phosphate scaffolds. Acta Biomaterialia. 2016;31:401-11 . [7] Gentleman E, Fredholm YC, Jell G, Lotfibakhshaiesh N, O'Donnell MD, Hill RG, et al. The effects of strontium-substituted bioactive glasses on osteoblasts and osteoclasts in vitro. Biomaterials. 2010;31:3949-56 . [8] Zhang W, Shen Y, Pan H, Lin K, Liu X, Darvell BW, et al. Effects of strontium in modified biomaterials. Acta Biomater. 2011;7:800-8. [9] Boda SK, Thrivikraman G, Panigrahy B, Sarma DD, Basu B. Competing Roles of Substrate Composition, Microstructure, and Sustained Strontium Release in Directing Osteogenic Differentiation of hMSCs. ACS Appl Mater Interfaces. 2016. [10] Yusa K, Yamamoto O, Takano H, Fukuda M, Iino M. Zinc-modified titanium surface enhances osteoblast differentiation of dental pulp stem cells in vitro. Sci Rep-Uk. 2016;6. [11] Ito A, Otsuka M, Kawamura H, Ikeuchi M, Ohgushi H, Sogo Y, et al. Zinc-containing tricalcium phosphate and related materials for promoting bone formation. Current Applied Physics. 2005;5:402-6. [12] Wang T, Zhang JC, Chen Y, Xiao PG, Yang MS. Effect of zinc ion on the osteogenic and adipogenic differentiation of mouse primary bone marrow stromal cells and the adipocytic trans-differentiation of mouse primary osteoblasts. Journal of Trace Elements in Medicine and Biology. 2007;21:84-91. [13] Seo HJ, Cho YE, Kim T, Shin HI, Kwun IS. Zinc may increase bone formation through stimulating cell proliferation, alkaline phosphatase activity and collagen synthesis in osteoblastic MC3T3-E1 cells. Nutr Res Pract. 2010;4:356 -61. [14] Jin SJ, Ren L, Yang K. Bio-Functional Cu Containing Biomaterials: a New Way to Enhance Bio-Adaption of Biomaterials. Journal of Materials Science & Technology. 2016;32:835-9. [15] Wu CT, Zhou YH, Xu MC, Han PP, Chen L, Chang J, et al. Copper-containing mesoporous bioactive glass scaffolds with multifunctional properties of angiogenesis capacity, osteostimulation and antibacterial activity. Biomaterials. 2013;34:422 -33. [16] Sen CK, Khanna S, Venojarvi M, Trikha P, Ellison EC, Hunt TK, et al. Copper-induced vascular endothelial growth factor expression and wound healing. Am J Physiol-Heart C. 2002;282:H1821-H7 . [17] Shi MC, Chen ZT, Farnaghi S, Friis T, Mao XL, Xiao Y, et al. Copper-doped mesoporous silica nanospheres, a promising immunomodulatory agent for inducing osteogenesis. Acta Biomaterialia. 2016;30:334-44.
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圖1A至1D：圖1A至1D描述了利用各種濃度的不同金屬離子所培養的人類間質幹細胞（human mescenchymal stem cell，hMSCs）的細胞存活率研究結果。圖1A描述了用鎂離子觀察到的典型結果。圖1B描述了用銅離子觀察到的典型結果。圖1C描述了用鋅離子觀察到的典型結果。圖1D描述了用鍶離子觀察到的典型結果。
圖2A及2B：圖2A及2B描述了用各種金屬離子所培養的人類間質幹細胞（hMSCs）其鹼性磷酸酶（alkaline phosphatase，ALP）經標準化的活性。圖2A顯示了以特定濃度的鎂、鋅、銅及鍶離子的各種組合處理後，於第7天及第14天的典型結果。圖2B顯示了鎂離子在不同濃度及時間點的典型結果。
圖3：圖3描述了本發明提出的植入物在受試者的股骨內的位置。
圖4A及4B：圖4A及4B描述了使用含金屬離子的植入物進行體內骨生長研究的結果。圖4A顯示在各時間點從植入了指示植入物的股骨所獲得的典型的Micro-CT圖像。圖4B描述了與植入物相鄰的骨體積的變化百分比；相對於僅含鎂的植入物而言，含有鎂/鍶/銅/鋅的植入物在第3、4及8週時，其相鄰的骨質顯著增多的現象。
圖5A及5B：圖5A及5B描述了使用本揭露所提出的含有金屬離子（含有不同含量的鎂、鍶、銅及鋅）的植入物進行體內骨生長研究的結果。圖5A顯示在各個時間點從植入了指示植入物的股骨所獲得的典型的Micro-CT圖像。圖5B描述了與植入物相鄰的骨體積的變化百分比。
圖6A至6C：圖6A、6B及6C描述了使用具有不同相對量的鎂、鍶、銅及鋅離子的本發明提出的含金屬離子植入物進行體內骨生長、股骨彈性係數（elastic modulus）及股骨硬度（stiffness）的研究結果。圖6A顯示在各時間點與指示植入物組合物相鄰的皮質骨（cortical bone）體積百分比變化的典型結果。圖6B顯示植入了指示植入物組合物，以進行完整股骨的彈性係數測量的典型結果。圖6B顯示植入了指示植入物組合物，以進行完整股骨的硬度測量的典型結果。Figures 1A to 1D: Figures 1A to 1D depict the results of cell viability studies of human mescenchymal stem cells (hMSCs) cultured with various concentrations of different metal ions. Figure 1A depicts typical results observed with magnesium ions. Figure 1B depicts typical results observed with copper ions. Figure 1C depicts typical results observed with zinc ions. Figure 1D depicts typical results observed with strontium ions.
Figures 2A and 2B: Figures 2A and 2B depict the normalized alkaline phosphatase (ALP) activity of human mesenchymal stem cells (hMSCs) cultured with various metal ions. Figure 2A shows typical results on
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