- Email: [email protected]
Impacts of the N-terminal fragment analog of human parathyroid hormone on structure, composition and biomechanics of bone
European Journal of Pharmaceutical Sciences 47 (2012) 926–933
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps
Impacts of the N-terminal fragment analog of human parathyroid hormone on structure, composition and biomechanics of bone Wang Chunxiao a,b,c,⇑, Zhang Yu b,d, Liu Wentao b,e, Liu Jingjing b, Ye Jiahui b, Chen Qingmei b a
Branch of Marine Biopharmaceutical, College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China Laboratory of Minigene Pharmacy, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China c Institute of Marine Sciences, Shanghai Ocean University, Shanghai 201306, China d School of Pharmacy, Nanjing Medical University, Nanjing 210029, China e Department of Pharmacology, Nanjing Medical University, Nanjing 210029, China b
a r t i c l e
i n f o
Article history: Received 13 May 2012 Received in revised form 27 August 2012 Accepted 12 September 2012 Available online 6 October 2012 Keywords: N-terminal fragment analog of hPTH Osteoporosis Biomechanic property Histomorphologic property Anti-fracture capability
a b s t r a c t Osteoporosis is a skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, and it is a serious threat to human lives. We previously showed that the N-terminal peptide analog of human parathyroid hormone (Pro-Pro-PTH(1–34)) enhanced plasma calcium concentration. In this paper, we study the impact of PTH N-terminal fragment analog on the structure, component, and mechanical properties of the rat bones. Daily subcutaneous injections of Pro-Pro-hPTH (1–34) induces 26.5–32.8% increase in femur bone mineral density (BMD), 23.0–34.2% decrease the marrow cavity or increase in trabecular bone area. The peptide also increases 16.0–59.5%, 28.8–48.2% and 14.0–17.8% of bone components of calcium, phosphorus and collagen, respectively. In terms of mechanic properties, administration of the peptide elevates the bone rigidity by 45.4–76.6%, decreases the flexibility by 23.0–31.6%, and improves modulus of elasticity by 32.8–63.4%. The results suggest that Pro-Pro-hPTH (1–34) has a positive effect on bone growth and strength, and possesses anti-fracture capability, thus a potential candidate for the application for the treatment of osteoporosis. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Osteoporosis is a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture (Kanis and The WHO Study Group, 1994). It seriously threatens the physical health of the human and profoundly impacts the life quality of people who suffer from it. Since it is one of the most prevalent and rapidly growing diseases in the world, the development of anti-osteoporosis drugs becomes a hotspot of the world pharmaceutical industry. Traditional therapeutic drugs such as estrogen, calcitonin and bisphosphonate prevent bone loss and decrease the rate of fracture in postmenopausal women through inhibiting bone resorption, but they cannot help the bone tissue return to its normal density and strength. A great deal of clinical research shows that sequential treatment with intermitAbbreviations: cpm, count per minute, a unit of radioactivity; Fmc, percent area fraction of marrow cavity; Ftb, trabecular bone area fraction; hPTH, human parathyroid hormone; LP, low power field; OVXed, Ovarectomized; R2V, raster to vector conversion software; SEM, scanning electron microscopy; TPBFT, three-point bending flexural test. ⇑ Corresponding author at: Branch of Marine Biopharmaceutical, College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China. Tel.: +86 21 61900387; fax: +86 21 61900365. E-mail address: [email protected] (W. Chunxiao). 0928-0987/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejps.2012.09.013
tent low-dose human parathyroid hormone (PTH) enhanced bone formation, and the N-terminal portion of the peptide PTH(1–34) retains the full biologic activity of the intact peptide (1–84) (Chunxiao et al., 2011). Due to the effect of PTH and its analog on both prevention of osteolysis and promotion of bone growth and recovery, they are considered the first choice among the 2nd generation drugs for treating osteoporosis. Pure Pro-Pro-hPTH (1–34), a recombinant human parathyroid hormone (1–34) analog with N-terminal Pro-Pro extension was prepared in our lab by genetic engineering technique and showed significant plasma calcium-increasing activity (Chunxiao et al., 2007). In this paper, we use adult ovarectomized (OVX) and sexually matured female rat as an animal model to evaluate the therapeutic potential of the peptide Pro-Pro-hPTH (1–34). We found that the peptide elevated bone components, reduced bone marrow cavity, and improved bone biomechanical properties, thus showed its capability of preventing or treating osteoporosis.
2. Materials and methods 2.1. Ovarectomized rat Ten-week-old, virgin female Sprague Dawley rats (Clean animals, CL) were purchased from SHANGHAI SIPPR-BK LAB ANIMAL
W. Chunxiao et al. / European Journal of Pharmaceutical Sciences 47 (2012) 926–933
CO., LTD in China and raised until they were 3 month old in the Animal Care Facility of the Laboratory of Minigene Pharmacy, China Pharmaceutical University, Nanjing, China. The rats were fed commercial rat chow containing 20.0–25.0% protein, 5.0–10.0% fat, 3.0–5.0% crude fiber, 0.9–1.0% calcium, 0.6–0.8% phosphorus, 0.00005% vitamin D, and ad libitum. The rats were housed in a room maintained at 25 °C with a 12-h light/dark cycle. All animals were treated following guidelines of the Animal Care Center, China Pharmaceutical University. At age of 3 months, the rats were randomized into seven groups according to weight with at least 10 animals each. Five groups were subjected to bilateral ovariectomies by the dorsal approach (Waynforth and Flecknell, 1992), while two groups were subjected to sham surgery to exteriorize and replace the ovaries. For surgery, the animals were anesthetized by intraperitoneal injections of freshly prepared pentobarbital sodium (8 mg/mL, sterile saline vehicle of 0.15 M NaCl in distilled water) of 0.5 mL/100 g body weight. Soon after the operation, all animals received daily intramuscular injections of 6.4 104 U penicillin (dissolved in 0.2 mL normal saline solution) per rat for 7 days, and 25 mg streptomycin (dissolved in 0.15 mL normal saline) per rat for 4 days to prevent infection, with erythromycin ointment applied externally. All rats were left untreated for 98 days after the surgery, to allow for the development of severe osteopenia (Morley et al., 1997). Rat weight was measured every two weeks to assess weight changes. 2.2. Daily administration with Pro-Pro-hPTH (1–34) At the end of the 14th week, the rats were re-grouped by weight. The control animals were divided into two groups of the sham-baseline group (Sb) (n = 10) and the sham group (n = 11). Among the other animals that had undergone bilateral ovariectomies, 11 were selected as OVX baseline animals (Ob), 13 OVX animals (Os) received daily subcutaneous injections of comparable volumes of an acidic saline vehicle (0.15 M NaCl in distilled water containing 0.001 N HCl) for 16 weeks starting at the end of the 14th week after OVX, and 39 OVX animals (Op) received daily subcutaneous injections of geometric series 0.4, 0.6 or 0.9 nmol of Pro-Pro-hPTH (1–34)/100 g body weight (dissolved in the acidic saline vehicle mentioned above) for 16 weeks, as suggested by Whitfield’s work involving daily subcutaneous injections of 0.4 or 0.8 nmol/100 g of body weight of the two anabolic agents rhPTH(1–84) and PTH-(1–31)NH2 (Ostabolin) (Whitfield et al., 1997). The values of BMD were determined for the Sb and Ob groups at the end of the 14th week after the surgery and for the Os and Op groups at the end of the 16th week after the 1st subcutaneous injection, following the experimental design in Sogaard’s work (Sogaard et al., 1997). The rats were weighed and sacrificed on the next day of the determination of BMD to collect femurs and blood samples for later use. 2.3. Determination of bone mineral density Bone mineral density was measured at the femur by Dual Energy X-ray Absorptiometry (DEXA)(LUNARÒ Expert #1170). The rats were anesthetized with ether, kept face down and their limbs spreading, and body straight. The whole body of each rat was scanned. The BMD values of the whole right femur were automatically displayed and saved in the computer. During the scan analysis, the operator was blinded for the treatment and scan order, except for rat identity. 2.4. Animal sacrifice and sample acquisition A day after receiving the last dose, the rats were exsanguinated via the femoral vein, and all effluent blood was collected for subse-
927
quent measurement on turnover of serum biochemical markers. Bilateral femurs were disarticulated from the hip and knee joints, with removal of all soft tissue and excess muscle, and preserved for determination of bone biomechanical properties and histomorphologic variations (the right femurs), or bone chemical composition (Ca and collagen, the left femurs). The uteri were examined for evidence of atrophy. 2.5. Biomechanical testing The biomechanical properties of bone in OVXed rats were evaluated with the TPBFT at 20 °C in 15 min after the disarticulation. This test was adopted rather than the compression and tension experiments because most bone fractures happen in case of impact or stress and the irregular bone shape is easily handled by the TPBFT experiments. The dimensions of the outer major and minor axes of the cleanly scraped femurs were determined with a vernier caliper before the test, while the dimension of the inner major and minor axes and the thickness of the bone wall of the broken bones were determined after the test. An intact right femur was placed in a flexural test machine (2000 N, Instron, USA) on two supports under the ends of the bone, with a span of 20 mm. The load was gradually increased in direction of minor axis on the femur midpoint until the specimen was broken into two pieces. A sophisticated sensor and recorder system (SEU-HYP programmable data recorder, derived by the Institute of Mechanics, Southeast University) ensures that the microcomputer receives a continuous feed of data on the load and the displacement (the largest distances of the central line from the original position) of the femur. Using Grapher software, the load-deformation curves can be plotted automatically. The maximum load Fmax, stiffness EJ, maximum stress rbmax, maximum deflection dpmax, and modulus of elasticity E were calculated from the curves by using the following formulae from bending beam theory: (1) J = p(BH3 bh3)/64 (J: cross-sectional moment of inertia; B: outer major axis; H: outer minor axis; b: inner major axis; h: inner minor axis dimension); (2) rb = FmaxLH/8J (L: the span of the beam); and (3) E = FmaxL3/ 48J dpmax. 2.6. Microscopy and image analysis 2.6.1. Bone tissue preparation The two parts of the broken bone (right) obtained from the TPBFT were retrieved, with removal of all residual connective tissues. 2.6.2. Histologic evaluation For light microscopic examination, the distal end of the femur was carefully sectioned longitudinally, with both sides of the epiphysis preserved in order to obtain maximum information. All of the specimens were fixed in 10% formalin buffer, decalcified in formic acid, dehydrated in graded ethanol, embedded in paraffin wax, sectioned at 4 lm using a sledge microtome, and stained with hematoxylin and eosin (HE). The slides were examined by light microscopy at a 40 or 100 magnification and photomicrographs were taken. Four representatives of the homologous sections from each animal were selected for the test. The unused distal ends were preserved for phosphorus determination. 2.6.3. Morphometric analysis by SEM SEM was adopted here since it is easy to measure Fmc and Ftb in a SEM image (rather than a light microscopy image) by using R2V, to determine the morphometric change in the bone sample.
928
W. Chunxiao et al. / European Journal of Pharmaceutical Sciences 47 (2012) 926–933
2.6.3.1. Tissue preparation. For SEM analysis the proximal segment (the femoral head end) of right femur was fixed in a 4% glutaraldehyde solution in 0.1 M phosphate pH7.3 for several days, and then sectioned sagittally by a diamond blade saw. An undamaged section was selected from each animal, cleaned with water, immersed in 10% sodium hypochlorite for 6 h, ultrasonically cleaned in water for 15 min, dehydrated in graded ethanol, immersed in ether, dried in air, sputter coated with gold using an IB-3 Ion Coater, and finally viewed axially under a Scanning Electronic Microscope (SEM) (Akashi-Kaikyo SX-40, Japan), with an accelerating voltage of 20 kV. Six representative homologous sections (one from each animal) were selected for each group (Electron Microscopy Room, Analysis Center, China Pharmaceutical University). 2.6.3.2. Bone morphometric analysis. The trabecular diameters were measured at the thinnest site of each trabecula for each group separately. By using a scanner and R2V, the SEM images were first converted to digitized computer images and displayed on a computer monitor, then the void spaces (marrow cavity) were circled and the area was integrated to determine the bone marrow cavity size. The Ftb can be obtained from the Fmc. 2.7. Biochemical analysis 2.7.1. Serum biochemical testing 2.7.1.1. Blood samples. Venous blood collected by exsanguination was allowed to clot for 30 min at room temperature and then centrifuged at 1300 g for 10 min. Serum samples were then aliquoted and frozen at 20 °C before analysis.
were considered to be significant for P values between 0.01 and 0.05 (indicated by ()), very significant for P between 0.001 and 0.01 (), extremely significant for P < 0.001 () and non-significant for P > 0.05 (without any mark). 3. Results After examination, the data were deleted for altogether 11 animals with no evidence of atrophy of the uterus observed. In all the histograms below, OpL, OpM and OpH each represents for low, medium and high dose (0.4, 0.6 or 0.9 nmol of Pro-Pro-hPTH (1–34)/ 100 g body weight respectively). 3.1. Body weight Since mature assimilation of rats is greater than dissimilation, it is not surprising to observe body weight gain with age in the test. As shown in Fig. 1, ovariectomy (OVX) induced an elevation of body weight of rat, which was an estrogen deficiency-induced body-weight gain (Liu et al., 2006). Intermittent Pro-Pro-hPTH (1–34) administration can elevate body weight very significantly (P < 0.01) as compared to the sham-operated animals. However, the body weight was not affected so much (P > 0.05) in comparison with the vehicle-injected OVX rats (the body weight gain is not significant but dose-dependent). Hence, we speculate that the body weight increase is primarily caused by increase of bone weight. 3.2. Bone mass measurement
2.7.1.2. Measurement of biochemical markers of bone turnover. The serum Ca, P and alkaline phosphatase (ALPase) levels were determined with an automatic biochemical analyzer (Beckman SYNCHRON LX System) in the Clinical Analysis Unit (ZhongDa Hospital, Nanjing, Jiangsu, PR China). The indirect potentiometry, a timed rate method and a kinetic rate method were used to analyze the data. The serum osteocalcin (OC, bone gla protein, BGP) concentration was determined by RIA in ZhongDa Hospital, Nanjing, Jiangsu, PR China through a serum osteocalcin radioimmunoassay kit (Shanghai Biological Products Research Institute, Shanghai, China) using a SN-682B-3 radioimmuno counter according to the procedures described in the instruction manual, which was adapted from the method described before (Power et al., 1989; Simionescu et al., 1988).
Bone mass parameters (femoral mineral density, femoral dry weight, femoral Ca, femoral P and femoral collagenous hydroxyproline (cHyp) content were measured in femur to evaluate the osteogenic effect of Pro-Pro-hPTH (1–34). As shown in Fig. 2, there was no significant difference in the most parameters for almost all groups of control rats with different treatments, except for that femoral mineral density of age matched Sham is significantly elevated (P < 0.05). Intermittent Pro-Pro-hPTH (1–34) administration elevated femoral mineral density, femoral dry weight, femoral Ca, P and cHyp content very significantly, with the exception of Ca content of femur treated with low dose Pro-Pro-hPTH (1–34).
2.7.2. Bone biochemical testing Left lateral femurs harvested from rats were baked at 80 °C until constant weights were achieved (about 48 h). The dried femurs were dissolved in 6 mol/L HCl at 108 °C for 16 h. The digestive juices were filtered, diluted for subsequent analysis of hydroxyproline content and for calcium determination, by the hydroxyproline assay kit or a calcium assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The distal ends of the right femurs were handled according to the manufacturer’s instruction contained in inorganic phosphorus assay kit (Nanjing Jiancheng).
Bone geometric parameters (femoral length; femoral outer major and minor axis lengths) were measured. ‘Anabolic drugs build bigger bone, not just denser’ (Deal and Gideon, 2003). As shown in Fig. 3, there was no significant differences between the OVX rats and the age matched Sham rats (P > 0.05) both 14 and 30 weeks after OVX, and between the sham-operated rats 30 weeks after OVX and the Sb rats 14 weeks after OVX (P > 0.05), except for a
3.3. Bone geometric properties
2.8. Statistical analysis The data of the animals whose uteri did not show any sign of atrophy were deleted. The data were analyzed by an Excel based (Microsoft Office) two-tailed paired student T test. After statistical analysis, all values determined were expressed as average ± standard deviation (bars). In comparison to control values (a. Sb as control; b. Sham as control; and c. Os as control. The control groups are marked as Sba, Shamb and Osc, respectively, for notice), differences
Fig. 1. Effects of Pro-Pro-hPTH (1–34) on the body weight of rats (n P 9).
W. Chunxiao et al. / European Journal of Pharmaceutical Sciences 47 (2012) 926–933
929
Fig. 3. Effects of Pro-Pro-hPTH (1–34) on geometric measurements of the femur(mm) (n P 9). (A). femoral length; (B). femoral outer major axis length and outer minor axis length.
3.4. Biomechanics findings
Fig. 2. Effects of Pro-Pro-hPTH (1–34) on bone mass parameters (measured on femur). (A) Femoral mineral density (n = 8) (g/cm2). (B) Femoral dry weight (n P 9) (g). (C) Collagen per femur (n P 9) (results are expressed as milligram (mg) of hydroxyproline per femur). (D) Femoral calcium (n = 11) (mmol/L) and (E) Femoral Phosphorus (n P 10) (mg).
significant increase in femoral length (P < 0.01). Intermittent ProPro-hPTH (1–34) administration increased femoral length in OVX rats significantly (P < 0.001, as compared with the vehicle group (36.73 ± 0.70)), but not significantly the femoral outer major and minor axis lengths (P > 0.05, as compared with the vehicle groups (4.01 ± 0.10 and 2.89 ± 0.06)).
The effects of Pro-Pro-hPTH (1–34) on biomechanics was evaluated using the right femur. The following biomechanical parameters were either measured or calculated. As shown in Fig. 4, there was a significant decrease in maximum stress (P < 0.05) of the femurs of Ob rats compared with that of Sb rats, but no significant difference in maximum load, stiffness, maximum deflection, and modulus of elasticity (P > 0.05). When the femur of sham-operated rats 30 weeks after OVX is compared with that of Sb rats 14 weeks after OVX, there was a very significant increase in maximum load (P < 0.01), a more significant increase in the stiffness, modulus of elasticity (P < 0.001), but no significant decrease in maximum stress (P > 0.05), and a significant decrease in the maximum deflection (P < 0.001). OVX induced osteopenia, as reflected by significant decrease in maximum load (P < 0.05) 30 weeks after OVX, whereas Pro-ProhPTH (1–34) administration significantly increases maximum load, stiffness, and modulus of elasticity of the femur. Compared with the vehicle-treated group, there was a significant decrease of maximum deflection in the rats at low and medium doses of Pro-Pro-hPTH (1–34) (P < 0.05), but no significant decrease in at high dose (P > 0.05). And no significant decrease (P > 0.05) in all Pro-Pro-hPTH (1–34) administration groups when compared with sham group. Thus, OVX decreases the maximum load, stiffness, maximum stress, elastic modulus, but increases the maximum deflection; Pro-Pro-hPTH (1–34) reverses these effects in various degrees. The effect of Pro-Pro-hPTH (1–34) on modulus of elasticity is dose dependent.
3.5. Histological findings (the distal section, some figures are shown in the Supplementary Materials) After stained with HE, the light microphotos showed that the bone ‘‘ossification’’ observed in Ob rats was not as obvious as that in the Sb group, accompanied by reduced osteoblast proliferation, absence of cancellous bone or sparsely distributed trabeculae, in-
930
W. Chunxiao et al. / European Journal of Pharmaceutical Sciences 47 (2012) 926–933
and narrower than those of the Sham group and no osteoclasts were found. No mineralized nodule was observed in the bone marrow of the rats for both the control and the treated groups. 3.6. Bone histomorphologic and histomorphometric findings (the proximal section) 3.6.1. Characteristics description of SEM sections The cancellous bone specimens, taken from the caput femoris of each experimental group, are composed of a network of multilayer arch bridges (bony trabeculae), separated by a labyrinth of interconnected pores (marrow cavities) inside (Fig. 5A). The small marrow cavities with ovate or spindle-shape in Sb or Sham group, which are encircled by the arch-bridge-shaped bone spicules or trabeculae interwoven, interconnected to form large bone marrows, in all the samples from the vehicle-injected animals, Ob or Os. In some regions, the microarchitecture of bone was disrupted, and the trabeculae slender, formed an irregular meshwork. The bridging part (the middle part) of the trabecula was significantly attenuated, and there is loss of horizontal trabeculae. As the microdamage accumulated, the bone surface appears to be damaged and very rugged (figures not shown). The average trabecular diameter (Ut) in Ob or Os group was significantly decreased in comparison with the Sb or Sham group. The significant marrow cavity expansion shown in Fig. 5A-Ob, Fig. 5A-Os was due to OVX-induced trabecular resorption and attenuation. In the groups treated with various doses of Pro-Pro-hPTH (1–34), the marrow cavities appear to have spindle/oval shape and smaller size as the dose increased, the bone surface is smoother and the average Ut were significantly increased, when compared with the Os group. Those are indications that this peptide has effect on trabecular bone formation(Individual marrow cavities seem longer and narrower compared with the Sham group, those are traces left which tell stories about a previous osteoporosis event and are indirect evidences which show that Pro-Pro-hPTH (1–34) is an anabolic agent rather than an antiresorptive agent).
Fig. 4. Effects of Pro-Pro-hPTH (1–34) on biomechanics of the femur (n P 9). (A) maximum load (N); (B). stiffness (N/mm); (C) maximum stress (MPa); (D) dpmax, maximum deflection (mm); and (E) modulus of elasticity (MPa).
creased marrow cavity, and fewer and indistinct cement lines (2–3 Osteoclasts/LP). The trabeculae number and thickness were reduced in the Os group, in comparison with the Sham group. In some areas there are dispersed fragments of the cancellous bone, with some osteoblasts adjacent to them. Rare osteoclasts are found in the Os group, whereas no osteoclasts were observed in the Sham group. Intermittent administration with various doses Pro-Pro-hPTH (1–34) led to various degrees of increase in trabeculae number and thickness, reduction of marrow cavity, significant improvement in bone microarchitecture, and strikingly increased number of live osteoblasts(an evidence which show that Pro-Pro-hPTH (1–34) is an anabolic agent). However, individual marrow cavities within trabecular bone of the femurs of these groups appear to be longer
3.6.2. Statistical results The average Ut and Ftb measured in six representative sections of each group were shown in Fig. 5B and C, respectively. In Fig. 5C, the lower and upper rectangulars represent Ftb, Fmc, respectively. When the Fmc fraction increases, Ftb decreases, and vice versa. It is obvious that there was a significant decrease in average Ut and Ftb of the OVX rats compared with the age matched Sham rats (P < 0.001) both 14 and 30 weeks after OVX. There was no significant difference in average Ut and Ftb between the Sham rats 30 weeks after OVX and the Sb rats 14 weeks after OVX (P > 0.05). Intermittent administration with various doses ProPro-hPTH (1–34) resulted in significant elevation in average Ut and Ftb of the rat femur (P < 0.001), and the increase is proportional to the Pro-Pro-hPTH (1–34) dosage. 3.7. Impact of Pro-Pro-hPTH (1–34) on biochemical parameters 3.7.1. Serum biochemical parameters Serum components were assayed by an auto-biochemical analyzer (SYNCHRON LX system) or other corresponding kits, including serum Ca, P, ALPase, and OC. 3.7.1.1. OC determination. The serum OC concentrations were calculated according to the regression equation log IT(Y) = A + B log(X). A = 1.081235, B = 1.140803, R = .999096. As shown in Fig. 6A, there was a significant increase of OC in the rats at medium dose of Pro-Pro-hPTH (1–34), when either compared with the vehicle group (P < 0.05) or the sham-operated rats (P < 0.05). However,
W. Chunxiao et al. / European Journal of Pharmaceutical Sciences 47 (2012) 926–933
931
Fig. 6. Effects of Pro-Pro-hPTH (1–34) on biochemical parameters (n P 9). (A), serum OC. (B), serum ALPase. (C), serum calcium and serum phosphorus.
Fig. 5. Evaluation of the effect of Pro-Pro-hPTH (1–34) under a scanning electron microscope (n = 6). Six representative homologous sections (one from each animal) were selected for each group. (A), Representative images of the proximal ends of the femurs under a scanning electron microscope (70). (B), A comparison of average trabecula diameters (lm) (measured at the thinnest portion of the trabeculae). (C), A comparison of the percent area fraction of marrow cavity and the trabecular bone area fraction.
no significant difference (P > 0.05) in OC was found between other two groups. 3.7.1.2. ALPase level. The serum ALPase activity was measured by monitoring the changes in absorbance at 405 nm (IFCC-DGKCh). As shown in Fig. 6B, there was no significant difference (P > 0.05) in serum ALPase activity between the OVX and sham-operated animals 14 weeks after OVX. However, a significant decrease in serum ALPase concentration was observed for the Sham animals 30 weeks after OVX in comparison with the Sb group 14 weeks after OVX (P < 0.05). There was a significant increase in serum ALPase level of the vehicle-treated group compared with the age matched sham (P < 0.05), which reflect the increased turnover associated with bone destruction of menopause, a phenomenon happened to osteoporotic women (Jenkins, 2001; Wei et al., 2010) and osteoporosis model of rats (Xu et al., 2004), since the elevation of bone-ALP make total ALP level elevated several times in patients with bone disease (Day et al., 1992). It indicates that a typical ovariectomy-
induced osteoporosis model has been established. The serum ALPase level was not significantly affected by treatment of Pro-Pro-hPTH (1–34) when compared with the vehicle-injected OVX animals (P > 0.05). Whereas progressively greater increases in ALPase level resulting from increasing dose and the high dose group showed significant elevation (P < 0.05) in comparison with the sham group, which maybe another evidence that the treatment with Pro-Pro-hPTH (1–34) is an anabolic therapy, just like a gradually increasing dose of antiresorptive therapy could bring about progressively greater decreases in ALPase level (Jenkins, 2001).
3.7.1.3. Serum Ca and P level. As shown in Fig. 6C, there was no significant difference (P > 0.05) in serum Ca concentrations between any rats with the exception of low dose Pro-Pro-hPTH (1–34) when compared with sham. However, a significant decrease of serum P concentrations was observed for the sham rats 30 weeks after OVX in comparison with those 14 weeks after (Sb) (P < 0.001), but a significant (P < 0.05 and P < 0.001) increases of serum P concentrations in the medium and high dose groups when compared with sham rats. However, no significant difference (P > 0.05) in serum P concentrations was observed between other two groups. A great increase (though not significant) was observed for the Os group in comparison with the age matched Sham, a phenomenon happened to osteoporotic women, which was probably due to decreased estrogen production and elevated growth hormone levels (Wei et al., 2010).
932
W. Chunxiao et al. / European Journal of Pharmaceutical Sciences 47 (2012) 926–933
4. Discussion The accurate biomechanical properties such as maximum stress and Young’s modulus can only be fully evaluated if the structural parameters (i.e., maximum load and stiffness) are corrected for changes in geometric properties of the femur midshaft (Ederveen et al., 2001). In our study, the Young’s modulus increased significantly (P < 0.05) or very significantly (P < 0.01) for the Pro-ProhPTH (1–34) administration groups, indicating improvement of biomechanical properties by our compounds, although the increase of maximum stress was not significant (P > 0.05). We speculate that a great decrease in maximum deflection may prevent an increase of maximum stress, although there was a significant increase of maximum load and stiffness of the rats of the Pro-ProhPTH (1–34) groups. Osteocalcin, also known as a bone gamma-carboxyglutamicacid-containing protein (Bone Gla-protein, BGP), is a noncollagen protein in bone and is thought to play a role in body’s metabolic regulation and is pro-osteoblastic, or bone-building by nature. It is also implicated in bone mineralization and Ca2+ homeostasis. In many studies, OC is used as a preliminary biomarker for the effectiveness of a drug on bone formation. One percent of bone matrix is composed of OC, which is produced by mature osteoblast and odontoblast. OC is quickly cleared by kidney after it is released into blood circulation. The half-life of circulating OC is only around 4–5 min. Thus serum OC should reflect the latest status of OC synthesis, bone formation and bone turnover over the entire skeleton. As shown in Fig. 6A, the PTH fragment did influence the synthesis of OC. And on different stages of osteoblast differentiation, Pro-ProhPTH (1–34) showed different effects. A 16 week administration of the medium dose of Pro-Pro-hPTH (1–34) induced conversion of pre-osteoblasts to osteoblasts in the rats, while the latter, in turn, produced more OC as a regulator to inhibit the activity of the cells themselves (Lee et al., 2007; Ivaska et al., 2004; Kronenberg, 1997; Bodine and Komm, 1999). Osteoblasts are rich in the enzyme ALPase, which plays a major role in the formation of the mineral deposits in the matrix (Capra and Conti, 2009). During matrix maturation, the cell surface enzyme ALP may become solubilized and circulate in the blood. Levels of total serum ALP (tsALP) can then be measured in the laboratory and is indicative of the amount of bone formation occurring. From the result shown in Fig. 6B, we can deduce that the osteoblasts in the Sham rats 30 weeks after OVX are not as active as that of the Sb rats 14 weeks after OVX, according to the corresponding ALPase levels. This is in accordance with the fact that bone formation decreases with age. However, the osteoblasts did not significantly change the activity in the rats that were given various doses of Pro-Pro-hPTH (1–34). The osteoblasts should be more active in the vehicle group than that in the sham group since its serum ALPase activity was significantly higher. Since all animals in the vehicle group developed severe osteoporosis(Fig. 6A-Os), we speculate that the osteoporosis may have stimulated more osteoblasts, which in turn synthesize and secrete more collagen fibers (Eroschenko, 2008). That may explain slightly higher content of collagen in this group. The inorganic material, consisting mainly of hydroxyapaptite crystals (Ca10(PO4)6(OH)2), is deposited within the osteoid and helps to resist compressive strains placed on bone. The organic matrix resists bending and tensile strains. The increase in femoral Ca content in the medium and high dose groups was significant. The increase in femoral P content in the high dose group is more significant than that in the low and medium dose groups, whereas the increase extent in collagen content seems become lesser as the dose increases. The above facts suggest that the dose difference led to alterations in the ratio of organic to inorganic content of bone. In practical terms, as the dose increases, the increase in inor-
ganic Ca, P in femora is more significant, whereas organic collagen did not show this potential. We speculate that those inorganic Ca, P possess a competitive advantage over its rivals organic collagen for occupying the micro-spaces when the dose increased. The direction of these changes is in accordance with the observed changes in biomechanical results, in which, the stiffness increased appreciably for Pro-Pro-hPTH (1–34) injected animals, and tend to increase more when the dose increased. The above results suggest that the anabolic effect of 16-weeks treatment with higher dose Pro-Pro-hPTH (1–34) on bone formation in severe postmenopausal osteoporosis mainly manifests as enhancing the mineralization of the newly formed trabeculae. Progressively greater increases in ALPase brought about by a gradually increasing dose shown in Fig. 6B may be a more direct reason for this phenomenon (Capra and Conti, 2009). With more inorganic Ca, P composing the bone, the bone stiffness and strength would be elevated, while softness reduced. That is why the maximum deflection recovered to normal levels (comparable to sham animals) from elevated levels in the OVX state. However, in some cases, excessive mineralization may increase bone brittleness, according to the result of recent reports (Hernandez, 2008). It is also observed that femur stiffness in control rats 30 weeks after OVX is significantly elevated, whereas the deflection at maximum is significantly reduced when compared with those 14 weeks after OVX. This is in accordance with the fact that the proportion of fluid and of organic material decreases with age, whereas that of inorganic material increases and the bones become stiff and brittle. An elevated level of Ca was not observed in serum samples 24 h after the final injection, so it is speculated that serum Ca returned to normal level quickly or gradually, without induction of chronic hypercalcemia. Physiological level of estrogen promotes the growth plate closure, whereas OVX, which can bring about an estrogen-depleted state, can often accelerate longitudinal bone growth (Sogaard et al., 1997; Chagin et al., 2004). That is the reason why femur lengths in Os and Ob groups are longer than those in the Sham and Sb groups in our animal tests. The bones of the Pro-Pro-hPTH (1–34) injected groups are characterized by relatively small marrow cavities and thick bone trabeculae (Fig. 5A-Op), consistent with virtually no osteoclast in those groups(as indicated in the histological findings). The lackness may be achieved through inhibiting the osteoclasts recruiting to the bone surface, preventing the osteoclasts from binding to the bone, or shortening the lifespan of osteoclasts, presumably by inducing apoptosis (Clarkson, 2011). All these results reflect a taste of ‘balance’, a delicate balance of endocrine system. Besides bone density, the factors that reduce fractures may include drug effects on bone turnover, microarchitecture, quality, and geometry, and local cellular effects. It is known that [ProPro-hPTH(1–34)], as a prodrug of hPTH(1–34), would undergo in vivo cleavage of first Pro-Pro to give hPTH(1–34), using the same mechanism of action as hPTH(1–34) (Chunxiao et al., 2007). [ProPro-hPTH(1–34)] may prevent fractures not only by increasing bone mass: it may also change the geometry of bone, just as an actual drug hPTH(1–34) does(Uusi-Rasi et al., 2002; Zanchetta et al., 2003). Some researchers had studied Forteo, an actual drug PTH(1– 34) of rDNA origin, and obtained the peak time and the vanishing period of plasma concentrations to be approximately 30 min and 3 h. And it was deduced that given in this manner, Forteo stimulates new bone formation by stimulating osteoblast activity to a greater extent than osteoclast activity (Deal and Gideon, 2003). Certainly a prodrug would bring about some slight or big difference on the features mentioned above. For this reason, it would be interesting to do some thorough research on the comparison of this pro-
W. Chunxiao et al. / European Journal of Pharmaceutical Sciences 47 (2012) 926–933
drug with hPTH(1–34) to understand the role of segment Pro-Pro in stability, availability and pharmacokinetics in the future. Acknowledgements This work was supported by a funding from National Natural Science of China (Grant No. 30270298), the National High Technology ‘‘863’’ Programs of China (No. 2002 AA217031-2), Leading Academic Discipline Project of Shanghai Municipal Education Commission (Project No. J50704) and SHOU Project 05-207. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ejps.2012.09.013. References Bodine, P.V.N., Komm, B.S., 1999. Evidence that conditionally immortalized human osteoblasts express an osteocalcin receptor. Bone 25 (5), 535–543. Capra, P., Conti, B., 2009. The role of Bone Morphogenetic Proteins (BMPs) in bone tissue engineering: a mini review. Scientifica Acta 3 (1), 25–32. Chagin, A., Lindberg, M.K., Andersson, N., Moverare, S., Gustafsson, J.A., Vendahl, L.S., Ohlsson, C., 2004. Estrogen receptor-b inhibits skeletal growth and has the capacity to mediate growth plate fusion in female mice. J. Bone Miner. Res. 19 (1), 72–77. Chunxiao, W., Jingjing, L., Yire, X., Min, D., Zhaohui, W., Gaofu, Q., Xiangchun, S., Xuejun, W., Jie, W., Taiming, L., 2007. Study on preparation and activity of a novel recombinant human parathyroid hormone (1–34) analog with N-terminal Pro-Pro extension. Regul. Pept. 141, 35–43. Chunxiao, W., Jingjing, L., Yire, X., Jingning, L., Kai, K., Liang, S., Yi, L., Rasco, B., 2011. Biosynthesis of a novel recombinant peptide derived from hPTH(1–34). Protein Expr. Purif. 79 (1), 156–163. Clarkson, P., 2011. Dem bones, dem important bones. The Science Creative Quarterly (6). Day, A.P., Saward, S., Royle, C.M., Mayne, P.D., 1992. Evaluation of two new methods for routine measurement of alkaline phosphatase isoenzymes. J. Clin. Pathol. 45 (1), 68–71. Deal, C., Gideon, J., 2003. Recombinant human PTH 1–34 (Forteo): An anabolic drug for osteoporosis. Clev. Clin. J. Med. 70 (7), 585–601. Ederveen, A.G.H., Spanjers, C.P.M., Quaijtaal, J.H.M., Kloosterboer, H.J., 2001. Effect of 16 months of treatment with tibolone on bone mass, turnover, and biomechanical quality in mature ovariectomized rats. J. Bone Miner. Res. 16 (9), 1674–1681. Eroschenko, V.P., 2008. Atlas of histology with functional correlations, 11th ed. Lippincott Williams & Wilkins, Baltimore.
933
Hernandez, C.J., 2008. How can bone turnover modify bone strength independent of bone mass? Bone 42 (6), 1014–1020. Ivaska, K.K., Hentunen, T.A., Vääräniemi, J., Ylipahkala, H., Pettersson, K., Väänänen, H.K., 2004. Release of intact and fragmented osteocalcin molecules from bone matrix during bone resorption in vitro. J. Biol. Chem. 279 (18), 18361–18369. Jenkins, D.K., 2001. Bone alkaline phosphatase, a serum bone turnover assay: usefulness in managing postmenopausal women receiving therapy to prevent or treat osteoporosis. Review series, pp. 1–8.. Kanis, J.A., The WHO Study Group, 1994. Assessment of fracture risk and its application to screening for postmenopausal osteoporosis: synopsis of a WHO report. Osteoporosis Int. 4, 368–381. Kronenberg, H.M., 1997. Editorial: parathyroid hormone and osteocalcin—when friends become strangers. Endocrinology 138 (8), 3083–3084. Lee, N.K., Sowa, H., Hinoi, E., Ferron, M., Ahn, J.D., Confavreux, C., Dacquin, R., Mee, P.J., McKee, M.D., Jung, D.Y., Zhang, Z., Kim, J.K., Mauvais-Jarvis, F., Ducy, P., Karsenty, G., 2007. Endocrine regulation of energy metabolism by the skeleton. Cell 130 (3), 456–469. Liu, X., Sawauchi, H., Ogawa, H., Kishida, T., Ebihara, K., 2006. Retrograded tapioca starch prevents ovarian hormone deficiency-induced hypercholesterolemia. J. Nutr. Sci. Vitaminol. 52 (2), 134–141. Morley, P., Whitfield, J.F., Willick, G.E., 1997. Anabolic effects of parathyroid hormone on bone. TEM 8 (6), 225–231. Power, M.J., Gosling, J.P., Fottrell, P.F., 1989. Radioimmunoassay of osteocalcin with polyclonal and monoclonal antibodies. Clin. Chem. 35 (7), 1408–1415. Simionescu, L., Dumitriu, L., Dimitriu, V., Zamfir-Grigorescu, D., Câmpean, G., Dumitriu, E., Balmesß, E., Simescu, M., Gudovan, E., Ursu, H., 1988. The serum osteocalcin levels in patients with thyroid diseases. Endocrinologie 26 (1), 27– 33. Sogaard, C.H., Mosekilde, L., Thomsen, J.S., Richards, A., McOsker, J.E., 1997. A comparison of the effects of two anabolic agents (Fluoride and PTH) on ash density and bone strength assessed in an osteopenic rat model. Bone 20 (5), 439–449. Uusi-Rasi, K., Beck, T.J., Oreskovic, T.L., Sato, M., Bogado, C.E., Zanchetta, J.R., 2002. Teriparatide (rhPTH 1–34) improves the structural geometry of the hip [abstract]. J. Bone Miner. Res. 17 (suppl 1), S208. Waynforth, H.B., Flecknell, P.A., 1992. Specific surgical operations,, .. Experimental and surgical techniques in the rat, second ed. Academic Press, London, pp. 276277. Wei, W., Wu, X., Li, Y., 2010. Experimental Methodology of Pharmacology, fourth ed. People‘s Medical Publishing House, Beijing (in Chinese). Whitfield, J.F., Morley, P., Willick, G.E., Ross, V., MacLean, S., Barbier, J.R., Isaacs, R.J., Ohannessian-Barry, L., 1997. Comparison of the ability of recombinant human parathyroid hormone, rhPTH-(1–84), and hPTH-(1–31)NH2 to stimulate femoral trabecular bone growth in ovariectomized rats. Calcified Tissue Int. 60 (1), 26–29. Xu, Z., Xiao, D., Yu, Z., Lu, X., Li, R., 2004. Expression of IL-6, BGP and B-ALP in osteoporosis model of rat. J. Xinxiang Med. Coll. 21 (4), 258–260 (in Chinese). Zanchetta, J.R., Bogado, C.E., Ferretti, J.L., Wang, O., Wilson, M.G., Sato, M., Gaich, G.A., Dalsky, G.P., Myers, S.L., 2003. Effects of teriparatide [recombinant human parathyroid hormone (1–34)] on cortical bone in postmenopausal women with osteoporosis. J. Bone Miner. Res. 18 (3), 539–543.
Contents lists available at SciVerse ScienceDirect
European Journal of Pharmaceutical Sciences journal homepage: www.elsevier.com/locate/ejps
Impacts of the N-terminal fragment analog of human parathyroid hormone on structure, composition and biomechanics of bone Wang Chunxiao a,b,c,⇑, Zhang Yu b,d, Liu Wentao b,e, Liu Jingjing b, Ye Jiahui b, Chen Qingmei b a
Branch of Marine Biopharmaceutical, College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China Laboratory of Minigene Pharmacy, School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China c Institute of Marine Sciences, Shanghai Ocean University, Shanghai 201306, China d School of Pharmacy, Nanjing Medical University, Nanjing 210029, China e Department of Pharmacology, Nanjing Medical University, Nanjing 210029, China b
a r t i c l e
i n f o
Article history: Received 13 May 2012 Received in revised form 27 August 2012 Accepted 12 September 2012 Available online 6 October 2012 Keywords: N-terminal fragment analog of hPTH Osteoporosis Biomechanic property Histomorphologic property Anti-fracture capability
a b s t r a c t Osteoporosis is a skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, and it is a serious threat to human lives. We previously showed that the N-terminal peptide analog of human parathyroid hormone (Pro-Pro-PTH(1–34)) enhanced plasma calcium concentration. In this paper, we study the impact of PTH N-terminal fragment analog on the structure, component, and mechanical properties of the rat bones. Daily subcutaneous injections of Pro-Pro-hPTH (1–34) induces 26.5–32.8% increase in femur bone mineral density (BMD), 23.0–34.2% decrease the marrow cavity or increase in trabecular bone area. The peptide also increases 16.0–59.5%, 28.8–48.2% and 14.0–17.8% of bone components of calcium, phosphorus and collagen, respectively. In terms of mechanic properties, administration of the peptide elevates the bone rigidity by 45.4–76.6%, decreases the flexibility by 23.0–31.6%, and improves modulus of elasticity by 32.8–63.4%. The results suggest that Pro-Pro-hPTH (1–34) has a positive effect on bone growth and strength, and possesses anti-fracture capability, thus a potential candidate for the application for the treatment of osteoporosis. Ó 2012 Elsevier B.V. All rights reserved.
1. Introduction Osteoporosis is a systemic skeletal disease characterized by low bone mass and microarchitectural deterioration of bone tissue, with a consequent increase in bone fragility and susceptibility to fracture (Kanis and The WHO Study Group, 1994). It seriously threatens the physical health of the human and profoundly impacts the life quality of people who suffer from it. Since it is one of the most prevalent and rapidly growing diseases in the world, the development of anti-osteoporosis drugs becomes a hotspot of the world pharmaceutical industry. Traditional therapeutic drugs such as estrogen, calcitonin and bisphosphonate prevent bone loss and decrease the rate of fracture in postmenopausal women through inhibiting bone resorption, but they cannot help the bone tissue return to its normal density and strength. A great deal of clinical research shows that sequential treatment with intermitAbbreviations: cpm, count per minute, a unit of radioactivity; Fmc, percent area fraction of marrow cavity; Ftb, trabecular bone area fraction; hPTH, human parathyroid hormone; LP, low power field; OVXed, Ovarectomized; R2V, raster to vector conversion software; SEM, scanning electron microscopy; TPBFT, three-point bending flexural test. ⇑ Corresponding author at: Branch of Marine Biopharmaceutical, College of Food Science and Technology, Shanghai Ocean University, Shanghai 201306, China. Tel.: +86 21 61900387; fax: +86 21 61900365. E-mail address: [email protected] (W. Chunxiao). 0928-0987/$ - see front matter Ó 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ejps.2012.09.013
tent low-dose human parathyroid hormone (PTH) enhanced bone formation, and the N-terminal portion of the peptide PTH(1–34) retains the full biologic activity of the intact peptide (1–84) (Chunxiao et al., 2011). Due to the effect of PTH and its analog on both prevention of osteolysis and promotion of bone growth and recovery, they are considered the first choice among the 2nd generation drugs for treating osteoporosis. Pure Pro-Pro-hPTH (1–34), a recombinant human parathyroid hormone (1–34) analog with N-terminal Pro-Pro extension was prepared in our lab by genetic engineering technique and showed significant plasma calcium-increasing activity (Chunxiao et al., 2007). In this paper, we use adult ovarectomized (OVX) and sexually matured female rat as an animal model to evaluate the therapeutic potential of the peptide Pro-Pro-hPTH (1–34). We found that the peptide elevated bone components, reduced bone marrow cavity, and improved bone biomechanical properties, thus showed its capability of preventing or treating osteoporosis.
2. Materials and methods 2.1. Ovarectomized rat Ten-week-old, virgin female Sprague Dawley rats (Clean animals, CL) were purchased from SHANGHAI SIPPR-BK LAB ANIMAL
W. Chunxiao et al. / European Journal of Pharmaceutical Sciences 47 (2012) 926–933
CO., LTD in China and raised until they were 3 month old in the Animal Care Facility of the Laboratory of Minigene Pharmacy, China Pharmaceutical University, Nanjing, China. The rats were fed commercial rat chow containing 20.0–25.0% protein, 5.0–10.0% fat, 3.0–5.0% crude fiber, 0.9–1.0% calcium, 0.6–0.8% phosphorus, 0.00005% vitamin D, and ad libitum. The rats were housed in a room maintained at 25 °C with a 12-h light/dark cycle. All animals were treated following guidelines of the Animal Care Center, China Pharmaceutical University. At age of 3 months, the rats were randomized into seven groups according to weight with at least 10 animals each. Five groups were subjected to bilateral ovariectomies by the dorsal approach (Waynforth and Flecknell, 1992), while two groups were subjected to sham surgery to exteriorize and replace the ovaries. For surgery, the animals were anesthetized by intraperitoneal injections of freshly prepared pentobarbital sodium (8 mg/mL, sterile saline vehicle of 0.15 M NaCl in distilled water) of 0.5 mL/100 g body weight. Soon after the operation, all animals received daily intramuscular injections of 6.4 104 U penicillin (dissolved in 0.2 mL normal saline solution) per rat for 7 days, and 25 mg streptomycin (dissolved in 0.15 mL normal saline) per rat for 4 days to prevent infection, with erythromycin ointment applied externally. All rats were left untreated for 98 days after the surgery, to allow for the development of severe osteopenia (Morley et al., 1997). Rat weight was measured every two weeks to assess weight changes. 2.2. Daily administration with Pro-Pro-hPTH (1–34) At the end of the 14th week, the rats were re-grouped by weight. The control animals were divided into two groups of the sham-baseline group (Sb) (n = 10) and the sham group (n = 11). Among the other animals that had undergone bilateral ovariectomies, 11 were selected as OVX baseline animals (Ob), 13 OVX animals (Os) received daily subcutaneous injections of comparable volumes of an acidic saline vehicle (0.15 M NaCl in distilled water containing 0.001 N HCl) for 16 weeks starting at the end of the 14th week after OVX, and 39 OVX animals (Op) received daily subcutaneous injections of geometric series 0.4, 0.6 or 0.9 nmol of Pro-Pro-hPTH (1–34)/100 g body weight (dissolved in the acidic saline vehicle mentioned above) for 16 weeks, as suggested by Whitfield’s work involving daily subcutaneous injections of 0.4 or 0.8 nmol/100 g of body weight of the two anabolic agents rhPTH(1–84) and PTH-(1–31)NH2 (Ostabolin) (Whitfield et al., 1997). The values of BMD were determined for the Sb and Ob groups at the end of the 14th week after the surgery and for the Os and Op groups at the end of the 16th week after the 1st subcutaneous injection, following the experimental design in Sogaard’s work (Sogaard et al., 1997). The rats were weighed and sacrificed on the next day of the determination of BMD to collect femurs and blood samples for later use. 2.3. Determination of bone mineral density Bone mineral density was measured at the femur by Dual Energy X-ray Absorptiometry (DEXA)(LUNARÒ Expert #1170). The rats were anesthetized with ether, kept face down and their limbs spreading, and body straight. The whole body of each rat was scanned. The BMD values of the whole right femur were automatically displayed and saved in the computer. During the scan analysis, the operator was blinded for the treatment and scan order, except for rat identity. 2.4. Animal sacrifice and sample acquisition A day after receiving the last dose, the rats were exsanguinated via the femoral vein, and all effluent blood was collected for subse-
927
quent measurement on turnover of serum biochemical markers. Bilateral femurs were disarticulated from the hip and knee joints, with removal of all soft tissue and excess muscle, and preserved for determination of bone biomechanical properties and histomorphologic variations (the right femurs), or bone chemical composition (Ca and collagen, the left femurs). The uteri were examined for evidence of atrophy. 2.5. Biomechanical testing The biomechanical properties of bone in OVXed rats were evaluated with the TPBFT at 20 °C in 15 min after the disarticulation. This test was adopted rather than the compression and tension experiments because most bone fractures happen in case of impact or stress and the irregular bone shape is easily handled by the TPBFT experiments. The dimensions of the outer major and minor axes of the cleanly scraped femurs were determined with a vernier caliper before the test, while the dimension of the inner major and minor axes and the thickness of the bone wall of the broken bones were determined after the test. An intact right femur was placed in a flexural test machine (2000 N, Instron, USA) on two supports under the ends of the bone, with a span of 20 mm. The load was gradually increased in direction of minor axis on the femur midpoint until the specimen was broken into two pieces. A sophisticated sensor and recorder system (SEU-HYP programmable data recorder, derived by the Institute of Mechanics, Southeast University) ensures that the microcomputer receives a continuous feed of data on the load and the displacement (the largest distances of the central line from the original position) of the femur. Using Grapher software, the load-deformation curves can be plotted automatically. The maximum load Fmax, stiffness EJ, maximum stress rbmax, maximum deflection dpmax, and modulus of elasticity E were calculated from the curves by using the following formulae from bending beam theory: (1) J = p(BH3 bh3)/64 (J: cross-sectional moment of inertia; B: outer major axis; H: outer minor axis; b: inner major axis; h: inner minor axis dimension); (2) rb = FmaxLH/8J (L: the span of the beam); and (3) E = FmaxL3/ 48J dpmax. 2.6. Microscopy and image analysis 2.6.1. Bone tissue preparation The two parts of the broken bone (right) obtained from the TPBFT were retrieved, with removal of all residual connective tissues. 2.6.2. Histologic evaluation For light microscopic examination, the distal end of the femur was carefully sectioned longitudinally, with both sides of the epiphysis preserved in order to obtain maximum information. All of the specimens were fixed in 10% formalin buffer, decalcified in formic acid, dehydrated in graded ethanol, embedded in paraffin wax, sectioned at 4 lm using a sledge microtome, and stained with hematoxylin and eosin (HE). The slides were examined by light microscopy at a 40 or 100 magnification and photomicrographs were taken. Four representatives of the homologous sections from each animal were selected for the test. The unused distal ends were preserved for phosphorus determination. 2.6.3. Morphometric analysis by SEM SEM was adopted here since it is easy to measure Fmc and Ftb in a SEM image (rather than a light microscopy image) by using R2V, to determine the morphometric change in the bone sample.
928
W. Chunxiao et al. / European Journal of Pharmaceutical Sciences 47 (2012) 926–933
2.6.3.1. Tissue preparation. For SEM analysis the proximal segment (the femoral head end) of right femur was fixed in a 4% glutaraldehyde solution in 0.1 M phosphate pH7.3 for several days, and then sectioned sagittally by a diamond blade saw. An undamaged section was selected from each animal, cleaned with water, immersed in 10% sodium hypochlorite for 6 h, ultrasonically cleaned in water for 15 min, dehydrated in graded ethanol, immersed in ether, dried in air, sputter coated with gold using an IB-3 Ion Coater, and finally viewed axially under a Scanning Electronic Microscope (SEM) (Akashi-Kaikyo SX-40, Japan), with an accelerating voltage of 20 kV. Six representative homologous sections (one from each animal) were selected for each group (Electron Microscopy Room, Analysis Center, China Pharmaceutical University). 2.6.3.2. Bone morphometric analysis. The trabecular diameters were measured at the thinnest site of each trabecula for each group separately. By using a scanner and R2V, the SEM images were first converted to digitized computer images and displayed on a computer monitor, then the void spaces (marrow cavity) were circled and the area was integrated to determine the bone marrow cavity size. The Ftb can be obtained from the Fmc. 2.7. Biochemical analysis 2.7.1. Serum biochemical testing 2.7.1.1. Blood samples. Venous blood collected by exsanguination was allowed to clot for 30 min at room temperature and then centrifuged at 1300 g for 10 min. Serum samples were then aliquoted and frozen at 20 °C before analysis.
were considered to be significant for P values between 0.01 and 0.05 (indicated by ()), very significant for P between 0.001 and 0.01 (), extremely significant for P < 0.001 () and non-significant for P > 0.05 (without any mark). 3. Results After examination, the data were deleted for altogether 11 animals with no evidence of atrophy of the uterus observed. In all the histograms below, OpL, OpM and OpH each represents for low, medium and high dose (0.4, 0.6 or 0.9 nmol of Pro-Pro-hPTH (1–34)/ 100 g body weight respectively). 3.1. Body weight Since mature assimilation of rats is greater than dissimilation, it is not surprising to observe body weight gain with age in the test. As shown in Fig. 1, ovariectomy (OVX) induced an elevation of body weight of rat, which was an estrogen deficiency-induced body-weight gain (Liu et al., 2006). Intermittent Pro-Pro-hPTH (1–34) administration can elevate body weight very significantly (P < 0.01) as compared to the sham-operated animals. However, the body weight was not affected so much (P > 0.05) in comparison with the vehicle-injected OVX rats (the body weight gain is not significant but dose-dependent). Hence, we speculate that the body weight increase is primarily caused by increase of bone weight. 3.2. Bone mass measurement
2.7.1.2. Measurement of biochemical markers of bone turnover. The serum Ca, P and alkaline phosphatase (ALPase) levels were determined with an automatic biochemical analyzer (Beckman SYNCHRON LX System) in the Clinical Analysis Unit (ZhongDa Hospital, Nanjing, Jiangsu, PR China). The indirect potentiometry, a timed rate method and a kinetic rate method were used to analyze the data. The serum osteocalcin (OC, bone gla protein, BGP) concentration was determined by RIA in ZhongDa Hospital, Nanjing, Jiangsu, PR China through a serum osteocalcin radioimmunoassay kit (Shanghai Biological Products Research Institute, Shanghai, China) using a SN-682B-3 radioimmuno counter according to the procedures described in the instruction manual, which was adapted from the method described before (Power et al., 1989; Simionescu et al., 1988).
Bone mass parameters (femoral mineral density, femoral dry weight, femoral Ca, femoral P and femoral collagenous hydroxyproline (cHyp) content were measured in femur to evaluate the osteogenic effect of Pro-Pro-hPTH (1–34). As shown in Fig. 2, there was no significant difference in the most parameters for almost all groups of control rats with different treatments, except for that femoral mineral density of age matched Sham is significantly elevated (P < 0.05). Intermittent Pro-Pro-hPTH (1–34) administration elevated femoral mineral density, femoral dry weight, femoral Ca, P and cHyp content very significantly, with the exception of Ca content of femur treated with low dose Pro-Pro-hPTH (1–34).
2.7.2. Bone biochemical testing Left lateral femurs harvested from rats were baked at 80 °C until constant weights were achieved (about 48 h). The dried femurs were dissolved in 6 mol/L HCl at 108 °C for 16 h. The digestive juices were filtered, diluted for subsequent analysis of hydroxyproline content and for calcium determination, by the hydroxyproline assay kit or a calcium assay kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The distal ends of the right femurs were handled according to the manufacturer’s instruction contained in inorganic phosphorus assay kit (Nanjing Jiancheng).
Bone geometric parameters (femoral length; femoral outer major and minor axis lengths) were measured. ‘Anabolic drugs build bigger bone, not just denser’ (Deal and Gideon, 2003). As shown in Fig. 3, there was no significant differences between the OVX rats and the age matched Sham rats (P > 0.05) both 14 and 30 weeks after OVX, and between the sham-operated rats 30 weeks after OVX and the Sb rats 14 weeks after OVX (P > 0.05), except for a
3.3. Bone geometric properties
2.8. Statistical analysis The data of the animals whose uteri did not show any sign of atrophy were deleted. The data were analyzed by an Excel based (Microsoft Office) two-tailed paired student T test. After statistical analysis, all values determined were expressed as average ± standard deviation (bars). In comparison to control values (a. Sb as control; b. Sham as control; and c. Os as control. The control groups are marked as Sba, Shamb and Osc, respectively, for notice), differences
Fig. 1. Effects of Pro-Pro-hPTH (1–34) on the body weight of rats (n P 9).
W. Chunxiao et al. / European Journal of Pharmaceutical Sciences 47 (2012) 926–933
929
Fig. 3. Effects of Pro-Pro-hPTH (1–34) on geometric measurements of the femur(mm) (n P 9). (A). femoral length; (B). femoral outer major axis length and outer minor axis length.
3.4. Biomechanics findings
Fig. 2. Effects of Pro-Pro-hPTH (1–34) on bone mass parameters (measured on femur). (A) Femoral mineral density (n = 8) (g/cm2). (B) Femoral dry weight (n P 9) (g). (C) Collagen per femur (n P 9) (results are expressed as milligram (mg) of hydroxyproline per femur). (D) Femoral calcium (n = 11) (mmol/L) and (E) Femoral Phosphorus (n P 10) (mg).
significant increase in femoral length (P < 0.01). Intermittent ProPro-hPTH (1–34) administration increased femoral length in OVX rats significantly (P < 0.001, as compared with the vehicle group (36.73 ± 0.70)), but not significantly the femoral outer major and minor axis lengths (P > 0.05, as compared with the vehicle groups (4.01 ± 0.10 and 2.89 ± 0.06)).
The effects of Pro-Pro-hPTH (1–34) on biomechanics was evaluated using the right femur. The following biomechanical parameters were either measured or calculated. As shown in Fig. 4, there was a significant decrease in maximum stress (P < 0.05) of the femurs of Ob rats compared with that of Sb rats, but no significant difference in maximum load, stiffness, maximum deflection, and modulus of elasticity (P > 0.05). When the femur of sham-operated rats 30 weeks after OVX is compared with that of Sb rats 14 weeks after OVX, there was a very significant increase in maximum load (P < 0.01), a more significant increase in the stiffness, modulus of elasticity (P < 0.001), but no significant decrease in maximum stress (P > 0.05), and a significant decrease in the maximum deflection (P < 0.001). OVX induced osteopenia, as reflected by significant decrease in maximum load (P < 0.05) 30 weeks after OVX, whereas Pro-ProhPTH (1–34) administration significantly increases maximum load, stiffness, and modulus of elasticity of the femur. Compared with the vehicle-treated group, there was a significant decrease of maximum deflection in the rats at low and medium doses of Pro-Pro-hPTH (1–34) (P < 0.05), but no significant decrease in at high dose (P > 0.05). And no significant decrease (P > 0.05) in all Pro-Pro-hPTH (1–34) administration groups when compared with sham group. Thus, OVX decreases the maximum load, stiffness, maximum stress, elastic modulus, but increases the maximum deflection; Pro-Pro-hPTH (1–34) reverses these effects in various degrees. The effect of Pro-Pro-hPTH (1–34) on modulus of elasticity is dose dependent.
3.5. Histological findings (the distal section, some figures are shown in the Supplementary Materials) After stained with HE, the light microphotos showed that the bone ‘‘ossification’’ observed in Ob rats was not as obvious as that in the Sb group, accompanied by reduced osteoblast proliferation, absence of cancellous bone or sparsely distributed trabeculae, in-
930
W. Chunxiao et al. / European Journal of Pharmaceutical Sciences 47 (2012) 926–933
and narrower than those of the Sham group and no osteoclasts were found. No mineralized nodule was observed in the bone marrow of the rats for both the control and the treated groups. 3.6. Bone histomorphologic and histomorphometric findings (the proximal section) 3.6.1. Characteristics description of SEM sections The cancellous bone specimens, taken from the caput femoris of each experimental group, are composed of a network of multilayer arch bridges (bony trabeculae), separated by a labyrinth of interconnected pores (marrow cavities) inside (Fig. 5A). The small marrow cavities with ovate or spindle-shape in Sb or Sham group, which are encircled by the arch-bridge-shaped bone spicules or trabeculae interwoven, interconnected to form large bone marrows, in all the samples from the vehicle-injected animals, Ob or Os. In some regions, the microarchitecture of bone was disrupted, and the trabeculae slender, formed an irregular meshwork. The bridging part (the middle part) of the trabecula was significantly attenuated, and there is loss of horizontal trabeculae. As the microdamage accumulated, the bone surface appears to be damaged and very rugged (figures not shown). The average trabecular diameter (Ut) in Ob or Os group was significantly decreased in comparison with the Sb or Sham group. The significant marrow cavity expansion shown in Fig. 5A-Ob, Fig. 5A-Os was due to OVX-induced trabecular resorption and attenuation. In the groups treated with various doses of Pro-Pro-hPTH (1–34), the marrow cavities appear to have spindle/oval shape and smaller size as the dose increased, the bone surface is smoother and the average Ut were significantly increased, when compared with the Os group. Those are indications that this peptide has effect on trabecular bone formation(Individual marrow cavities seem longer and narrower compared with the Sham group, those are traces left which tell stories about a previous osteoporosis event and are indirect evidences which show that Pro-Pro-hPTH (1–34) is an anabolic agent rather than an antiresorptive agent).
Fig. 4. Effects of Pro-Pro-hPTH (1–34) on biomechanics of the femur (n P 9). (A) maximum load (N); (B). stiffness (N/mm); (C) maximum stress (MPa); (D) dpmax, maximum deflection (mm); and (E) modulus of elasticity (MPa).
creased marrow cavity, and fewer and indistinct cement lines (2–3 Osteoclasts/LP). The trabeculae number and thickness were reduced in the Os group, in comparison with the Sham group. In some areas there are dispersed fragments of the cancellous bone, with some osteoblasts adjacent to them. Rare osteoclasts are found in the Os group, whereas no osteoclasts were observed in the Sham group. Intermittent administration with various doses Pro-Pro-hPTH (1–34) led to various degrees of increase in trabeculae number and thickness, reduction of marrow cavity, significant improvement in bone microarchitecture, and strikingly increased number of live osteoblasts(an evidence which show that Pro-Pro-hPTH (1–34) is an anabolic agent). However, individual marrow cavities within trabecular bone of the femurs of these groups appear to be longer
3.6.2. Statistical results The average Ut and Ftb measured in six representative sections of each group were shown in Fig. 5B and C, respectively. In Fig. 5C, the lower and upper rectangulars represent Ftb, Fmc, respectively. When the Fmc fraction increases, Ftb decreases, and vice versa. It is obvious that there was a significant decrease in average Ut and Ftb of the OVX rats compared with the age matched Sham rats (P < 0.001) both 14 and 30 weeks after OVX. There was no significant difference in average Ut and Ftb between the Sham rats 30 weeks after OVX and the Sb rats 14 weeks after OVX (P > 0.05). Intermittent administration with various doses ProPro-hPTH (1–34) resulted in significant elevation in average Ut and Ftb of the rat femur (P < 0.001), and the increase is proportional to the Pro-Pro-hPTH (1–34) dosage. 3.7. Impact of Pro-Pro-hPTH (1–34) on biochemical parameters 3.7.1. Serum biochemical parameters Serum components were assayed by an auto-biochemical analyzer (SYNCHRON LX system) or other corresponding kits, including serum Ca, P, ALPase, and OC. 3.7.1.1. OC determination. The serum OC concentrations were calculated according to the regression equation log IT(Y) = A + B log(X). A = 1.081235, B = 1.140803, R = .999096. As shown in Fig. 6A, there was a significant increase of OC in the rats at medium dose of Pro-Pro-hPTH (1–34), when either compared with the vehicle group (P < 0.05) or the sham-operated rats (P < 0.05). However,
W. Chunxiao et al. / European Journal of Pharmaceutical Sciences 47 (2012) 926–933
931
Fig. 6. Effects of Pro-Pro-hPTH (1–34) on biochemical parameters (n P 9). (A), serum OC. (B), serum ALPase. (C), serum calcium and serum phosphorus.
Fig. 5. Evaluation of the effect of Pro-Pro-hPTH (1–34) under a scanning electron microscope (n = 6). Six representative homologous sections (one from each animal) were selected for each group. (A), Representative images of the proximal ends of the femurs under a scanning electron microscope (70). (B), A comparison of average trabecula diameters (lm) (measured at the thinnest portion of the trabeculae). (C), A comparison of the percent area fraction of marrow cavity and the trabecular bone area fraction.
no significant difference (P > 0.05) in OC was found between other two groups. 3.7.1.2. ALPase level. The serum ALPase activity was measured by monitoring the changes in absorbance at 405 nm (IFCC-DGKCh). As shown in Fig. 6B, there was no significant difference (P > 0.05) in serum ALPase activity between the OVX and sham-operated animals 14 weeks after OVX. However, a significant decrease in serum ALPase concentration was observed for the Sham animals 30 weeks after OVX in comparison with the Sb group 14 weeks after OVX (P < 0.05). There was a significant increase in serum ALPase level of the vehicle-treated group compared with the age matched sham (P < 0.05), which reflect the increased turnover associated with bone destruction of menopause, a phenomenon happened to osteoporotic women (Jenkins, 2001; Wei et al., 2010) and osteoporosis model of rats (Xu et al., 2004), since the elevation of bone-ALP make total ALP level elevated several times in patients with bone disease (Day et al., 1992). It indicates that a typical ovariectomy-
induced osteoporosis model has been established. The serum ALPase level was not significantly affected by treatment of Pro-Pro-hPTH (1–34) when compared with the vehicle-injected OVX animals (P > 0.05). Whereas progressively greater increases in ALPase level resulting from increasing dose and the high dose group showed significant elevation (P < 0.05) in comparison with the sham group, which maybe another evidence that the treatment with Pro-Pro-hPTH (1–34) is an anabolic therapy, just like a gradually increasing dose of antiresorptive therapy could bring about progressively greater decreases in ALPase level (Jenkins, 2001).
3.7.1.3. Serum Ca and P level. As shown in Fig. 6C, there was no significant difference (P > 0.05) in serum Ca concentrations between any rats with the exception of low dose Pro-Pro-hPTH (1–34) when compared with sham. However, a significant decrease of serum P concentrations was observed for the sham rats 30 weeks after OVX in comparison with those 14 weeks after (Sb) (P < 0.001), but a significant (P < 0.05 and P < 0.001) increases of serum P concentrations in the medium and high dose groups when compared with sham rats. However, no significant difference (P > 0.05) in serum P concentrations was observed between other two groups. A great increase (though not significant) was observed for the Os group in comparison with the age matched Sham, a phenomenon happened to osteoporotic women, which was probably due to decreased estrogen production and elevated growth hormone levels (Wei et al., 2010).
932
W. Chunxiao et al. / European Journal of Pharmaceutical Sciences 47 (2012) 926–933
4. Discussion The accurate biomechanical properties such as maximum stress and Young’s modulus can only be fully evaluated if the structural parameters (i.e., maximum load and stiffness) are corrected for changes in geometric properties of the femur midshaft (Ederveen et al., 2001). In our study, the Young’s modulus increased significantly (P < 0.05) or very significantly (P < 0.01) for the Pro-ProhPTH (1–34) administration groups, indicating improvement of biomechanical properties by our compounds, although the increase of maximum stress was not significant (P > 0.05). We speculate that a great decrease in maximum deflection may prevent an increase of maximum stress, although there was a significant increase of maximum load and stiffness of the rats of the Pro-ProhPTH (1–34) groups. Osteocalcin, also known as a bone gamma-carboxyglutamicacid-containing protein (Bone Gla-protein, BGP), is a noncollagen protein in bone and is thought to play a role in body’s metabolic regulation and is pro-osteoblastic, or bone-building by nature. It is also implicated in bone mineralization and Ca2+ homeostasis. In many studies, OC is used as a preliminary biomarker for the effectiveness of a drug on bone formation. One percent of bone matrix is composed of OC, which is produced by mature osteoblast and odontoblast. OC is quickly cleared by kidney after it is released into blood circulation. The half-life of circulating OC is only around 4–5 min. Thus serum OC should reflect the latest status of OC synthesis, bone formation and bone turnover over the entire skeleton. As shown in Fig. 6A, the PTH fragment did influence the synthesis of OC. And on different stages of osteoblast differentiation, Pro-ProhPTH (1–34) showed different effects. A 16 week administration of the medium dose of Pro-Pro-hPTH (1–34) induced conversion of pre-osteoblasts to osteoblasts in the rats, while the latter, in turn, produced more OC as a regulator to inhibit the activity of the cells themselves (Lee et al., 2007; Ivaska et al., 2004; Kronenberg, 1997; Bodine and Komm, 1999). Osteoblasts are rich in the enzyme ALPase, which plays a major role in the formation of the mineral deposits in the matrix (Capra and Conti, 2009). During matrix maturation, the cell surface enzyme ALP may become solubilized and circulate in the blood. Levels of total serum ALP (tsALP) can then be measured in the laboratory and is indicative of the amount of bone formation occurring. From the result shown in Fig. 6B, we can deduce that the osteoblasts in the Sham rats 30 weeks after OVX are not as active as that of the Sb rats 14 weeks after OVX, according to the corresponding ALPase levels. This is in accordance with the fact that bone formation decreases with age. However, the osteoblasts did not significantly change the activity in the rats that were given various doses of Pro-Pro-hPTH (1–34). The osteoblasts should be more active in the vehicle group than that in the sham group since its serum ALPase activity was significantly higher. Since all animals in the vehicle group developed severe osteoporosis(Fig. 6A-Os), we speculate that the osteoporosis may have stimulated more osteoblasts, which in turn synthesize and secrete more collagen fibers (Eroschenko, 2008). That may explain slightly higher content of collagen in this group. The inorganic material, consisting mainly of hydroxyapaptite crystals (Ca10(PO4)6(OH)2), is deposited within the osteoid and helps to resist compressive strains placed on bone. The organic matrix resists bending and tensile strains. The increase in femoral Ca content in the medium and high dose groups was significant. The increase in femoral P content in the high dose group is more significant than that in the low and medium dose groups, whereas the increase extent in collagen content seems become lesser as the dose increases. The above facts suggest that the dose difference led to alterations in the ratio of organic to inorganic content of bone. In practical terms, as the dose increases, the increase in inor-
ganic Ca, P in femora is more significant, whereas organic collagen did not show this potential. We speculate that those inorganic Ca, P possess a competitive advantage over its rivals organic collagen for occupying the micro-spaces when the dose increased. The direction of these changes is in accordance with the observed changes in biomechanical results, in which, the stiffness increased appreciably for Pro-Pro-hPTH (1–34) injected animals, and tend to increase more when the dose increased. The above results suggest that the anabolic effect of 16-weeks treatment with higher dose Pro-Pro-hPTH (1–34) on bone formation in severe postmenopausal osteoporosis mainly manifests as enhancing the mineralization of the newly formed trabeculae. Progressively greater increases in ALPase brought about by a gradually increasing dose shown in Fig. 6B may be a more direct reason for this phenomenon (Capra and Conti, 2009). With more inorganic Ca, P composing the bone, the bone stiffness and strength would be elevated, while softness reduced. That is why the maximum deflection recovered to normal levels (comparable to sham animals) from elevated levels in the OVX state. However, in some cases, excessive mineralization may increase bone brittleness, according to the result of recent reports (Hernandez, 2008). It is also observed that femur stiffness in control rats 30 weeks after OVX is significantly elevated, whereas the deflection at maximum is significantly reduced when compared with those 14 weeks after OVX. This is in accordance with the fact that the proportion of fluid and of organic material decreases with age, whereas that of inorganic material increases and the bones become stiff and brittle. An elevated level of Ca was not observed in serum samples 24 h after the final injection, so it is speculated that serum Ca returned to normal level quickly or gradually, without induction of chronic hypercalcemia. Physiological level of estrogen promotes the growth plate closure, whereas OVX, which can bring about an estrogen-depleted state, can often accelerate longitudinal bone growth (Sogaard et al., 1997; Chagin et al., 2004). That is the reason why femur lengths in Os and Ob groups are longer than those in the Sham and Sb groups in our animal tests. The bones of the Pro-Pro-hPTH (1–34) injected groups are characterized by relatively small marrow cavities and thick bone trabeculae (Fig. 5A-Op), consistent with virtually no osteoclast in those groups(as indicated in the histological findings). The lackness may be achieved through inhibiting the osteoclasts recruiting to the bone surface, preventing the osteoclasts from binding to the bone, or shortening the lifespan of osteoclasts, presumably by inducing apoptosis (Clarkson, 2011). All these results reflect a taste of ‘balance’, a delicate balance of endocrine system. Besides bone density, the factors that reduce fractures may include drug effects on bone turnover, microarchitecture, quality, and geometry, and local cellular effects. It is known that [ProPro-hPTH(1–34)], as a prodrug of hPTH(1–34), would undergo in vivo cleavage of first Pro-Pro to give hPTH(1–34), using the same mechanism of action as hPTH(1–34) (Chunxiao et al., 2007). [ProPro-hPTH(1–34)] may prevent fractures not only by increasing bone mass: it may also change the geometry of bone, just as an actual drug hPTH(1–34) does(Uusi-Rasi et al., 2002; Zanchetta et al., 2003). Some researchers had studied Forteo, an actual drug PTH(1– 34) of rDNA origin, and obtained the peak time and the vanishing period of plasma concentrations to be approximately 30 min and 3 h. And it was deduced that given in this manner, Forteo stimulates new bone formation by stimulating osteoblast activity to a greater extent than osteoclast activity (Deal and Gideon, 2003). Certainly a prodrug would bring about some slight or big difference on the features mentioned above. For this reason, it would be interesting to do some thorough research on the comparison of this pro-
W. Chunxiao et al. / European Journal of Pharmaceutical Sciences 47 (2012) 926–933
drug with hPTH(1–34) to understand the role of segment Pro-Pro in stability, availability and pharmacokinetics in the future. Acknowledgements This work was supported by a funding from National Natural Science of China (Grant No. 30270298), the National High Technology ‘‘863’’ Programs of China (No. 2002 AA217031-2), Leading Academic Discipline Project of Shanghai Municipal Education Commission (Project No. J50704) and SHOU Project 05-207. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.ejps.2012.09.013. References Bodine, P.V.N., Komm, B.S., 1999. Evidence that conditionally immortalized human osteoblasts express an osteocalcin receptor. Bone 25 (5), 535–543. Capra, P., Conti, B., 2009. The role of Bone Morphogenetic Proteins (BMPs) in bone tissue engineering: a mini review. Scientifica Acta 3 (1), 25–32. Chagin, A., Lindberg, M.K., Andersson, N., Moverare, S., Gustafsson, J.A., Vendahl, L.S., Ohlsson, C., 2004. Estrogen receptor-b inhibits skeletal growth and has the capacity to mediate growth plate fusion in female mice. J. Bone Miner. Res. 19 (1), 72–77. Chunxiao, W., Jingjing, L., Yire, X., Min, D., Zhaohui, W., Gaofu, Q., Xiangchun, S., Xuejun, W., Jie, W., Taiming, L., 2007. Study on preparation and activity of a novel recombinant human parathyroid hormone (1–34) analog with N-terminal Pro-Pro extension. Regul. Pept. 141, 35–43. Chunxiao, W., Jingjing, L., Yire, X., Jingning, L., Kai, K., Liang, S., Yi, L., Rasco, B., 2011. Biosynthesis of a novel recombinant peptide derived from hPTH(1–34). Protein Expr. Purif. 79 (1), 156–163. Clarkson, P., 2011. Dem bones, dem important bones. The Science Creative Quarterly (6)
933
Hernandez, C.J., 2008. How can bone turnover modify bone strength independent of bone mass? Bone 42 (6), 1014–1020. Ivaska, K.K., Hentunen, T.A., Vääräniemi, J., Ylipahkala, H., Pettersson, K., Väänänen, H.K., 2004. Release of intact and fragmented osteocalcin molecules from bone matrix during bone resorption in vitro. J. Biol. Chem. 279 (18), 18361–18369. Jenkins, D.K., 2001. Bone alkaline phosphatase, a serum bone turnover assay: usefulness in managing postmenopausal women receiving therapy to prevent or treat osteoporosis. Review series, pp. 1–8.