Effects of sequential therapy with recombinant human parathyroid hormone(1-84) and anti-resorptive agents on bones in ovariectomized rats

Abstract

[한글]

골흡수 억제제는 현재 골다공증치료에 주로 쓰이고 있으나, 골흡수억제만으로는 치료에 한계가 있다. 유전자 재조합 인간부갑상선호르몬(1-34)나 (1-84)는 저용량으로 간헐적 투여시 이미 에스트로겐 결핍 백서 및 폐경기 골다공증 여성에서 그 골밀도 촉진 효과가 잘 알려져 있고 최근 미국 FDA에서 승인을 받아 향후 골다공증치료에 큰 전환점이 될 것으로 생각된다. 하지만, 아직까지는 피하주사를 해야하는 단점이 있고 장기간 투여시 백서모델에서 발생했던 골종양에 대한 문제점이 있어, 인간에서 실질적으로 장기간 투여가 쉽지는 않을 것으로 생각된다. 또한 인간부갑상선호르몬을 투여 중단시에는 증가했던 골밀도가 다시 가역적으로 떨어지는 것으로 보고되고 있어, 인간부갑상선호르몬을 일단 투여하는 동화기 후에 기존의 골흡수 억제제를 유지기에 투여함으로써 증가된 골강도를 유지하는 요법에 대한 연구가 필요할 것으로 생각되어 본 연구를 진행하였다. 또한 골다공증 치료평가시 골밀도의 변화보다도 더 중요한 것은 실제 얼마나 골절을 예방할 것인가에 대한 골질의 변화일 것이다. 따라서 본 연구에서는 골량 및 골질의 양측면에서 이를 평가하고자 하였다./5개월된 36마리의 Sprague-Dawley 백서 암컷을 난소절제술을 시행하고 5주간 골소실기간을 주었으며 그 이후부터 치료를 시작하였다. 우선 5주간 인간재조합 부갑상선호르몬(1-84)[rhPTH(1-84)] 100 ㎍/㎏/d으로 1주에 5일간 투여했다. 그리고 나서는 5주간 각기 다른 방법으로 순차적 치료를 시행하였다. 각 군을 살펴보면, 1) 난소절제군(OVX) (n=6), 2) Sham 수술군(SHAM) (n=6), 3) PTH 유지군(PTH-M) (n=6), 4) PTH 퇴거군 (PTH-W) (n=6), 5) PTH후 여성호르몬치료군(17β-estradiol [10 ㎍/d, SQ *5 days/wk]) (PTH-E) (n=6), 6) PTH후 zoledronate치료군(PTH-Z) [12.5 ㎍/㎏, SQ weekly] (n=6). 그래서 본 연구에서는 인간부갑상선호르몬 투여 및 각 골흡수 억제제의 순차적 치료가 기존 골밀도 기기(Hologic QDR 4500A, Waltham, MA)를 이용한 골량측정과 마이크로 CT(Skyscan, Antwerpen, Belgium)를 이용한 미세구조 분석을 보았으며, 생체역학 분석(Instron LTD, Buckinghamshire, England)을 통한 골질 평가를 동시에 진행하였다./DXA나 마이크로 CT로 분석한 골밀도와 골량 등을 보면 PTH-M과 PTH-Z군에서 OVX나 PTH-W군보다도 의미있게 높았고(p<0.05), PTH-E군의 경우는 난소절제군과 PTH-M이나 PTH-Z군의 중간정도의 수치를 보였다. 대퇴골 중간부위에서 PTH-M군에서만 피질골 두께가 의미있게 다른 군보다 두꺼웠으며(p=0.001), 요추부에서 피질골 두께는 군간에 차이를 보이지 않았다. 생체역학검사상 수질골과 피질골에서 다른 결과를 보였는데, 대퇴부에서는 Fu값이 OVX군보다는 높았지만 다른 군간에 차이가 없었다. 그러나, PTH-M이나 PTH-Z군에서 난소절제군이나 PTH-E군에 비해 Fu값이 44-47%만큼 높았고 PTH-W군보다도 높은 경향을 보였다./결론적으로, PTH를 5주간 투여하고 중지한 경우 증가된 골강도가 저하되지만, 순차적으로 zoledronate를 쓰면 부갑상선호르몬으로 증가된 골밀도를 유지하는데 보다 효과적임을 알 수가 있었다.

[영문]

Anti-resorptive agents against osteoporosis are quite limited in terms of increasing bone mass by themselves. Strong anabolic agents, such as human parathyroid hormone (hPTH), are expected to be helpful. However, as hPTH is only injectable and has the critical side effect of bone tumors with life-long administration in the rat, it would be practical to use PTH for the shortest possible duration for the maximal effect. To determine the effectiveness of the concept of lose, restore, maintain(LRM), PTH was used to increase the bone mass, then the respective anti-resorptive agents after PTH was sequentially administered for the maintenance.

Thirty six, twenty-week-old, virgin female Sprague-Dawley rats were used. A bilateral ovariectomy(ovx) was performed at 20 weeks of age, and a further 5 weeks allowed for the osteopenic changes in the bones to take place. Each treatment was started at 25th week and continued for 10 weeks with the following regimen; recombinant human PTH(1-84) [rhPTH(1-84)] 100 ㎍/㎏/d*5 days/wk, and then the respective sequential therapies for 5 weeks, groups were as follows ; 1) Ovariectomized rats (OVX) (n=6), 2) Sham operated rats (SHAM) (n=6), 3) OVX rats with PTH maintenance (PTH-M) (n=6), 4) OVX rats treated with PTH then withdrawn(PTH-W) (n=6), 5) PTH treated OVX rats then treated with 17β-estradiol [10 ㎍/d, SQ *5 days/wk] (PTH-E) (n=6), 6) PTH treated OVX rats then treated with zoledronate(PTH-Z) [12.5 ㎍/㎏, SQ weekly] (n=6). Bone mineral densities (BMD) of the right femora were measured by dual x-ray absorptiometry (DXA)(Hologic QDR 4500A, Waltham, MA). The microcomputed tomography (μCT), and software, used for the structural parameter of the 2nd lumbar vertebrae were from Skyscan (Antwerpen, Belgium). Three-point bending test of the femora and compressive tests of vertebrae were performed with a material testing machine (Instron LTD, Buckinghamshire, England).

The data on the bone quantity showed that the BMD, measured by DXA and most of the microstructural parameters, by μCT, were higher in the PTH-M and PTH-Z groups than in the OVX and PTH-W groups (p < 0.05). Measurement of the cortical thickness revealed only the PTH-M group to have a significant increase (p=0.001). The tests on bone quality showed somewhat different results in the cortical and trabecular rich sites. The ultimate force (Fu) at the midshaft of the femora were similar in the treated groups, and stronger than in the OVX group (p<0.05). However, in the vertebrae, the Fu were significantly higher, by about 44-47%, in the PTH-M and PTH-Z groups than in the OVX and PTH-E groups, showing a higher tendency than in the PTH-W group.

In conclusion, PTH withdrawal resulted in the loss of the acquired BMD, and the sequential therapy, with anti-resorptives, prevented further loss with the respective agents (17β-estradiol vs. zoledronate), showing different efficacies at the different bony sites. The zoledronate after rhPTH(1-84) as a sequential regimen was quite consistently effective.

Anti-resorptive agents against osteoporosis are quite limited in terms of increasing bone mass by themselves. Strong anabolic agents, such as human parathyroid hormone (hPTH), are expected to be helpful. However, as hPTH is only injectable and has the critical side effect of bone tumors with life-long administration in the rat, it would be practical to use PTH for the shortest possible duration for the maximal effect. To determine the effectiveness of the concept of lose, restore, maintain(LRM), PTH was used to increase the bone mass, then the respective anti-resorptive agents after PTH was sequentially administered for the maintenance.

Thirty six, twenty-week-old, virgin female Sprague-Dawley rats were used. A bilateral ovariectomy(ovx) was performed at 20 weeks of age, and a further 5 weeks allowed for the osteopenic changes in the bones to take place. Each treatment was started at 25th week and continued for 10 weeks with the following regimen; recombinant human PTH(1-84) [rhPTH(1-84)] 100 ㎍/㎏/d*5 days/wk, and then the respective sequential therapies for 5 weeks, groups were as follows ; 1) Ovariectomized rats (OVX) (n=6), 2) Sham operated rats (SHAM) (n=6), 3) OVX rats with PTH maintenance (PTH-M) (n=6), 4) OVX rats treated with PTH then withdrawn(PTH-W) (n=6), 5) PTH treated OVX rats then treated with 17β-estradiol [10 ㎍/d, SQ *5 days/wk] (PTH-E) (n=6), 6) PTH treated OVX rats then treated with zoledronate(PTH-Z) [12.5 ㎍/㎏, SQ weekly] (n=6). Bone mineral densities (BMD) of the right femora were measured by dual x-ray absorptiometry (DXA)(Hologic QDR 4500A, Waltham, MA). The microcomputed tomography (μCT), and software, used for the structural parameter of the 2nd lumbar vertebrae were from Skyscan (Antwerpen, Belgium). Three-point bending test of the femora and compressive tests of vertebrae were performed with a material testing machine (Instron LTD, Buckinghamshire, England).

The data on the bone quantity showed that the BMD, measured by DXA and most of the microstructural parameters, by μCT, were higher in the PTH-M and PTH-Z groups than in the OVX and PTH-W groups (p < 0.05). Measurement of the cortical thickness revealed only the PTH-M group to have a significant increase (p=0.001). The tests on bone quality showed somewhat different results in the cortical and trabecular rich sites. The ultimate force (Fu) at the midshaft of the femora were similar in the treated groups, and stronger than in the OVX group (p<0.05). However, in the vertebrae, the Fu were significantly higher, by about 44-47%, in the PTH-M and PTH-Z groups than in the OVX and PTH-E groups, showing a higher tendency than in the PTH-W group.

In conclusion, PTH withdrawal resulted in the loss of the acquired BMD, and the sequential therapy, with anti-resorptives, prevented further loss with the respective agents (17β-estradiol vs. zoledronate), showing different efficacies at the different bony sites. The zoledronate after rhPTH(1-84) as a sequential regimen was quite consistently effective.