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Pharmacokinetics of midkine with different N-terminal structures in rats
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Pharmacokinetics of Midkine with Different N-terminal Structures in Rats Qing Deng , Xiaolan Yu , Shaorong Deng , Hao Ye , Yang Zhang , Jingjing Li Ph.D. , Wei Han , Yan Yu Ph.D. PII: DOI: Reference:
S0928-0987(20)30090-7 https://doi.org/10.1016/j.ejps.2020.105301 PHASCI 105301
To appear in:
European Journal of Pharmaceutical Sciences
Received date: Revised date: Accepted date:
18 September 2019 9 January 2020 8 March 2020
Please cite this article as: Qing Deng , Xiaolan Yu , Shaorong Deng , Hao Ye , Yang Zhang , Jingjing Li Ph.D. , Wei Han , Yan Yu Ph.D. , Pharmacokinetics of Midkine with Different Nterminal Structures in Rats, European Journal of Pharmaceutical Sciences (2020), doi: https://doi.org/10.1016/j.ejps.2020.105301
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
Pharmacokinetics of Midkine with Different N-terminal Structures in Rats Qing Deng1,2, Xiaolan Yu1, Shaorong Deng2, Hao Ye2, Yang Zhang2, Jingjing Li2, Wei Han2, Yan Yu1. 1
Shanghai Municipality Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, NO.800, Dongchuan Road, Shanghai, 200240, China. 2 Laboratory of Regeneromics, School of Pharmacy, Shanghai Jiao Tong University, NO.800, Dongchuan Road, Shanghai, 200240, China. * Correspondence: Yan Yu, Ph.D. Tel.: +86-021-34204902; Fax: +86-021-34204902; E-mail address: [email protected]. Jingjing Li, Ph.D. Tel.: +86-021-34205760; Fax: +86-021-34205760; E-mail address: [email protected]. Abstract Midkine (MK) is a heparin-binding growth factor that functions in multiple physiological processes, making it a promising drug target for treating various diseases including osteoarthritis (OA). However, the lack of pharmacokinetic studies on MK limits further clinical research. As the Ndomain of MK protein appears to be more important for its stability, this study aimed to investigate the pharmacokinetic profiles of recombinant human (rh)MK with different structures at the Nterminus via different administration routes in rats and guinea pigs. A single intramuscular (IM) injection of 1 mg/kg rhMK with or without extended sequences at the N-terminus expressed by Escherichia coli or Pichia was administered to six male SD rats. rhMK concentrations in sequential tail blood samples were measured by ELISA. rhMK without extended N-terminal sequences expressed by Pichia had a greater area under the curve (AUC), slower clearance, and longer half-life in rats following a single IM injection than those of the other rhMK proteins. The AUC values for rhMK after IM and intra-articular (IA) administration were 1523.3 ± 35.2 h×ng/mL and 872.0 ± 36.1 h×ng/mL, whereas the apparent volumes of distribution (Vd/f) were 0.184 ± 0.067 L/kg and 11.6 ± 0.8 L/kg, respectively, suggesting that rhMK was distributed more locally after IA injection than after IM injection as Vd/f magnitude gives a general idea of extent distribution in the body and higher Vd/f represents more locally distribution. rhMK concentration in the articular cartilage was markedly higher than that in serum and reached the highest level at 3 days after a single IA injection in Hartley guinea pigs. As the dose increased from 10 to 50 mg/kg, the AUC increased in a greaterthan-dose-proportional manner, suggesting that rhMK exhibits non-linear pharmacokinetics in rats after a single IM injection in this dose range. These results indicated that the N-terminal structure and administration route have substantial effects on the pharmacokinetics of rhMK in rats. Furthermore, rhMK was maintained in articular cartilage with minimal diffusion into the blood following IA injection in Hartley guinea pigs, providing a foundation for clinical research on the use of rhMK for OA treatment via IA delivery. Keywords: midkine, pharmacokinetic, N-terminal, intramuscular, intraarticular
1
Introduction
Midkine (MK) is a heparin-binding growth factor; it belongs to a small protein family with pleiotrophin (Muramatsu, 2014). MK enhances the survival, migration, cytokine expression, differentiation, and other activities of target cells. MK has important effects on various physiological processes, such as development, reproduction, and repair. It is also involved in the pathogenesis of inflammatory and malignant diseases (Kadomatsu et al., 2013). With respect to its structure, MK includes two main domains: an N-terminal N-domain and a C-terminal C-domain. The two domains are relatively independent with no detectable interactions. The C-domain contributes to major MK functions including neurite-promoting activity. The N-domain appears to be more important for protein stability (Muramatsu, 2014). Furthermore, due to a difference in the cleavage of the signal sequence, there are two forms of MK, differing in the presence of two extended amino acids (valinealanine or other amino acids) at the N-terminus. Thus, alteration of the N-terminal sequence is of great interest for the production of MK with a longer half-life (Muramatsu, 2014). Different routes of administration may be used to achieve either systemic or local protein and peptide delivery. For small therapeutic molecules, the drug delivery routes include parenteral (intravenous, intramuscular, and subcutaneous), oral, nasal, ocular, transmucosal (buccal, vaginal, and rectal), and transdermal. However, the routes for the administration of proteins and peptides are limited owing to their large size and structure. Active ingredients must be delivered directly to the target site in a rapid, easy, and convenient manner (Tandel et al., 2011). IM administration of drugs in aqueous solution results in rapid absorption of the drug in most cases, and drug absorption is influenced by factors that alter blood flow to the muscle (Bardal et al., 2011). We previously demonstrated that MK has a significant repair effect in rat models of articular cartilage injury and promotes the proliferation of articular chondrocytes in vitro and in vivo through IM injection (Zhang et al., 2010). Therefore, MK might serve as a candidate drug for osteoarthritis (OA). Several drugs are used to treat OA via intraarticular (IA) administration (Yu and Hunter, 2016); thus, IA injection of MK is a potential therapeutic approach for OA. Pharmacokinetic (PK) studies are of great importance to pave the way for clinical research on MK. In this study, we evaluated the pharmacokinetics of an extended N-terminal sequence expressed in Escherichia coli or without the extended N-terminal sequence expressed in both E. coli or Pichia, and determined the rhMK protein with ideal pharmacokinetic behavior in rats for further research. We also compared the PK parameters for different administration routes. These results provide a basis for further clinical studies on MK for OA treatment. 2
Materials and methods
2.1
Animals
Six-week-old male Sprague–Dawley (SD) rats and male 6-month-old Hartley guinea pigs were used. All rats and guinea pigs were housed in a barrier facility with 2 ppm chlorinated water, autoclaved food, and bedding, under a 12-h light–dark cycle and 40% humidity. Animals were provided free access to food and water. The animals were fasted for 12 h prior to dosing and for the duration of the experiment. The experimental protocols were approved by the Animal Care and Use Committee of School of Pharmacy, Shanghai Jiao Tong University (Animal Ethic Approval Number#2017052). 2
2.2
Reagents
The sources of the materials used were as follows: Recombinant Human Midkine (rhMK) (Jiaochen General Regeneratives (Shanghai) Limited, Shanghai, China); Human Midkine DuoSet ELISA (Cat#332410, R&D, Minneapolis, MN, USA); BCA Assay Kit (Beyotime, China). 2.3
Study design
All rats or Hartley guinea pigs were weighed (six SD rats or six Hartley guinea pigs per group). At 0 min, 1 mg/kg or 10 mg/kg rhMK was given IM or IA. 100 μL blood samples were then obtained from each rat by tail blood sampling at 5 min, 10 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h post-injection. The blood was collected in tubes and centrifuged for 30 min at 3000 rpm to collect the serum. The serum samples were stored at –80°C until analysis. 2.4
Total protein extraction from articular cartilage
Cartilage from the femoral condyles of knee joints in Hartley guinea pigs was diced and stored in liquid nitrogen after a single IA injection of rhMK (1 mg/kg) at 0 min, 1 h, 1 d, 3 d, 6 d, 9 d, 12 d. Total protein from frozen tissue was extracted using Bead Ruptor 24 Elite (OMNI, USA) according to manufacturer’s instructions. After extraction, the processed tissue was kept on ice for 30 min and centrifuged for 15 min (14,000 × g, 4°C). The concentration of the extracted protein was measured using the BCA Assay Kit and the rhMK concentration in articular cartilage was measured using the Human Midkine DuoSet ELISA Kit. rhMK concentration was normalized to the total protein concentration from articular cartilage to obtain the relative rhMK concentration in articular cartilage. 2.5
Enzyme-linked immunosorbent assay (ELISA)
rhMK concentrations in blood samples were analyzed by Human Midkine DuoSet ELISA according to the manufacturer’s instructions. Briefly, 100 μL of standard or 10-fold diluted serum sample by blank rat serum was incubated in a 96-well microplate (pre-coated with goat anti-human MK antibody) for 2 h at room temperature (25°C). Following three washes, biotinylated goat anti-human MK antibody was added and incubated with captured MK for 2 h at room temperature (25°C). After three further washes, 100 μL of streptavidin-conjugated horseradish-peroxidase was added and allowed to react for 30 min in the dark. The plate was then washed and the substrate solution (1:1 mixture of H2O2 and tetramethylbenzidine) was added to the wells (100 μL per well) for a 20-min reaction. Finally, 1 mol/L H2SO4 (stop solution) was added (50 μL per well) and the absorbance of the wells was measured at 450 nm. The sample concentrations were then determined after generating a standard curve using a four-parameter logistic curve-fit method. 2.6
Statistical analysis
A two-compartment model was selected to obtain the PK parameters of rhMK in rats using WinNonlin3.3. AUC, Cmax, Tmax and k (the elimination constant rate) were calculated using the above software and following were computation methods of T1/2, CL/f and Vd/f: T1/2=0.693/k, CL/f=dose/AUC, Vd/f=CL/f×T1/2/0.693. Results are expressed as means ± standard deviation (SD). Statistically significant differences among treatment groups were determined using a two-tailed unpaired t-test in Graphpad. P < 0.05 indicated significant differences. 3
Results
3.1
PK profiles for three kinds of rhMK protein in rats after single intramuscular injection 3
To investigate the effects of an extended N-terminal region of MK on PK progression, we analyzed three kinds of rhMK protein in rats after a single IM injection. The total blood volume of rat weighed 200-250 g was 11 mL-17.5 mL according to the Guidance for Nonclinical Pharmacokinetics of Medicinal Products” issued by National Medical Products Administration (NMPA). We collected 100 μL blood sample from each rat at pointed time and the total collected volume during 24 h was 1 mL (less than 10% of total blood volume).The serum concentration-time curves for rhMK with or without extended N-terminal sequences expressed by E. coli or Pichia after single IM injection are shown in Figure 1. The results are shown in Figure 2 and Supplemental Table 1. The maximum serum concentrations (Cmax) of rhMK without the extended N-terminal sequences expressed by E. coli (E.coli rhMK (KKK)) (503.0 ± 67.7 ng/mL) and Pichia (Pichia rhMK (KKK)) (465.6.0 ± 12.9 ng/mL) were much greater than those of rhMK with extended N-terminal sequences expressed by E. coli (E.coli rhMK (VAKKK)) (198.1 ± 87.7 ng/mL). The Tmax for E. coli rhMK (KKK) and Pichia rhMK (KKK) was 0.9 ± 0.2 h and 1.0 ± 0.6 h, respectively, which was shorter than that for E.coli rhMK (VAKKK) (1.8 ± 0.5 h). The area under the curve (AUC) for Pichia rhMK (KKK) (1523.3 ± 35.2 h×ng/mL) was significantly higher than that of E. coli rhMK (KKK) (1278.4 ± 110.3 h×ng/mL, p = 0.0059) and E. coli rhMK (VAKKK) (861.8 ± 36.8h×ng/mL, p < 0.0001). However, the terminal half-lives (T1/2) of E. coli rhMK (VAKKK), E. coli rhMK (KKK), and Pichia rhMK (KKK) were 1.4 ± 0.4 h, 2.5 ± 1.1 h, and 2.4 ± 0.8 h, respectively, and did not differ significantly. The volume of distribution (Vd/f) and body clearance rate (CL/f) of Pichia rhMK (KKK) (0.184 ± 0.067 L/kg; 0.0537 ± 0.002 L/h/kg) were lowest among those for the three kinds of rhMK. These results support the use of Pichia rhMK (KKK) in subsequent research. 3.2
Comparison of PK behavior of rhMK following intramuscular or intraarticular administration in rats
The serum concentration-time curves for Pichia rhMK (KKK) via IM injection and IA injection are shown in Figure 3. The Cmax of Pichia rhMK (KKK) following IM delivery (465.6 ± 12.9 ng/mL) was significantly higher than that after IA injection (303.7 ± 33.3 ng/mL, p < 0.0001). The AUC and Tmax values for Pichia rhMK (KKK) during IM injection (1523.3 ± 35.2 h×ng/mL; 1.0 ± 0.6 h) were markedly higher than those for IA injection (872.0 ± 36.1 h×ng/mL, p < 0.0001 ; 0.5 ± 0.0 h, p = 0.0005), whereas the T1/2 values for Pichia rhMK (KKK) after administration by IM or IA routes were similar at 2.4 ± 0.8 h and 1.9 ± 0.1 h, respectively. However, the Vd/f and CL/f values for Pichia rhMK (KKK) after IM injection (0.184 ± 0.067 L/kg; 0.0537 ± 0.002 L/h/kg) were considerably lower than those of Pichia rhMK (KKK) after IA injection (11.6 ± 0.8 L/kg, p < 0.0001; 4.2 ± 0.2 L/h/kg, p < 0.0001) (Figure 4, Supplemental Table 2). 3.3
Pichia rhMK (KKK) storage in articular cartilage of Hartley guinea pigs after intraarticular injection
The serum rhMK concentration-time curve and articular cartilage rhMK concentration-time curve after IA injection are shown in Figure 5. The maximum rhMK serum concentration was detected at 10 min after dosing, followed by a rapid decrease. The articular cartilage rhMK concentration reached the highest level at 3 days after IA injection and a relatively high concentration was maintained for about 9 days. 3.4
Effect of dosage on rhMK PK profiles in rats
Serum PK parameters for Pichia rhMK (KKK) were evaluated after IM administration using doses of 10 mg/kg or 50 mg/kg, as summarized in Figures 6–7 and Supplemental Table 3. For Pichia rhMK (KKK) following IM injection with different doses, the increases in AUC and C max were dose4
dependent (10 mg/kg: 1298.4 ± 199.3 h×ng/mL < 50 mg/kg: 10139.4 ± 497.7 h×ng/mL, p < 0.0001; 10 mg/kg: 524.6 ± 198.9 ng/mL < 50 mg/kg: 3399.4 ± 769.2 ng/mL, p = 0.0033). However, T1/2 and Tmax values for Pichia rhMK (KKK) at doses of 10 mg/kg or 50 mg/kg did not differ significantly after IM injection. CL/f was decreased as the dose increased, from 0.039 L/h/kg at 10 mg/kg to 0.005 L/h/kg at 50 mg/kg (p=0.007). Vd/f ranged from 0.124 to 0.017 L/kg at the tested doses (p = 0.0006). 4
Discussion
We performed a PK analysis of rhMK with or without extended N-terminal sequences expressed by E. coli or Pichia in rats after IM or IA injection. An isoform with two additional amino acids (valine-alanine or other amino acids) at the N-terminus is present in MK preparations from different species due to a difference in the cleavage of the signal sequence. The N domain appears to be more important for the stability of MK. Therefore, to produce MK with a longer half-life, the extended form that has an additional VA at the N terminus, is of great interest. Limited proteolysis of MK by chymotrypsin is specifically inhibited by binding to heparin (Matsuda and Talukder, 1996) and MK with the extended N-terminus binds heparin more weakly compared to MK without the extended sequence (Muramatsu, 2014). In this study, we compared the main PK parameters of rhMK with or without extended N-terminal sequences expressed by E. coli and rhMK without an extended N-terminal sequence expressed by Pichia (Figures 1–2). The similar Cmax, T1/2, and Tmax values of E.coli rhMK(KKK) and Pichia rhMK(KKK) suggested that the different protein expression systems did not influence the rate of rhMK absorption in rats after a single IM injection. However, considerable differences in PK parameters between E. coli rhMK (KKK) and E. coli rhMK (VAKKK) suggested that the extended sequence at the N terminus regulated the absorption, distribution, and clearance of rhMK in rats after a single IM injection. rhMK without the extended N terminal sequence showed better absorption, as evidenced by the higher AUC and Cmax and lower Tmax. This kind of rhMK protein was distributed mainly in the circulation and displayed slower elimination, as indicated by lower Vd/f and CL/f values. Therefore, we used the rhMK protein without the extended N terminal sequence as our target for subsequent analyses. In a comparison of the main PK parameters for E. coli rhMK (KKK) and Pichia rhMK (KKK), we found that rhMK expressed by Pichia displayed better absorption and slower elimination, with a higher AUC and lower CL/f. Based on these results, rhMK without the extended N terminal sequence expressed by Pichia was found ideal for further pharmacokinetic research. Different administration routes affect PK progression for the same protein under identical experimental conditions (Persky, 2013). We previously found that rhMK is involved in promoting proliferation of rat chondrocytes in vivo and in vitro through IM injection (Zhang et al., 2010). Thus, we conducted a PK study of rhMK after a single IM or IA injection (Figures 3–4) and evaluated the articular cartilage rhMK concentration after IA delivery in rats (Supplemental Figure 4). The data suggested that less rhMK enters the circulation is due to a more local distribution after IA injection than after IM injection, as indicated by the lower AUC and C max and higher Vd/f as well as the storage of relative high concentration of rhMK in articular cartilage for almost 9 days. However, the terminal half-life of rhMK after IM injection and IA injection did not differ markedly. Furthermore, Hartley guinea pigs are widely used experimental animals for preclinical studies of OA (Little and Zaki, 2012), and thus, we evaluated the serum rhMK concentration and the relative rhMK concentration in articular cartilage following single IA injection in Hartley guinea pigs (Figure 5). rhMK was poorly absorbed into circulation, as indicated by the low C max of rhMK in the serum after IA injection. MK reached the highest concentration 3 days after injection and was maintained in the
5
articular cartilage for almost 9 days. These results illustrate that rhMK can be maintained in articular cartilage to exert its repair function. Some therapeutic protein drugs display non-linear PK behavior in humans (Zhou and Theil, 2015). To evaluate whether or not MK is one such protein drug, we performed a PK study of rhMK after IM injection with 10 mg/kg or 50 mg/kg (Figures 6–7). The results suggest that absorption increases with an increase in dosage after IM injection. However, we also found that the Vd/f and CL/f values for rhMK following IM injection decrease as the dose increases. This can potentially be explained by target-mediated drug disposition, which occurs when binding to the pharmacodynamic target structure and affects the PK dynamics of a drug compound, resulting in capacity-limited saturable processes. The consequence of these saturable processes, caused by the limited availability of enzymes, receptors, or other protein structures that interact with the drug, is non-linear PK behavior, including higher clearance and a larger apparent volume of distribution at lower doses (Zhou and Theil, 2015). Previously, we demonstrated that MK promotes the proliferation of chondrocytes by LRP1 mediated endocytosis (Deng, 2020), thus, LRP1 might be one of the targets interacting with MK to affect its PK behavior. In this study, we found that rhMK in rats after IM injection exhibits non-linear pharmacokinetic behavior; in particular, the dose-normalized concentration vs. time profiles at different dosages is non-superimposable for IM injection. Thus, the CL/f and Vd/f for rhMK were higher at lower doses following IM injection. Noriyuki Suzuki has demonstrated that lysosomal system and proteasomal system are involved in the degradation of endocytosed midkine in embryonic fibroblasts in vitro (Noriyuki, 2004). However, the elimination process of rhMK in rats or humans is still unclear and needs to be clarified. This is the first PK study of MK with different structures at the N-terminus administered through various injection routes. We obtained the following main results. 1) The extended form of the Nterminus of rhMK has a great effect on its pharmacokinetic characteristics and rhMK without extended N-terminal sequences expressed by Pichia displays the best PK behavior in rats after a single IM injection among the three rhMK proteins evaluated. 2) rhMK was maintained in articular cartilage and exhibited little diffusion into circulation after single IA administration in rats and guinea pigs. 3) rhMK exhibited non-linear PK behavior in rats after IM injection within a dose range of 10–50 mg/kg. The PK profiles of MK with different structures administered through different routes described in this study improve our understanding of the pharmacology of MK and provide a basis for its clinical use in mammals. 5
Abbreviations
Midkine (MK); intramuscular (IM); intraarticular (IA); terminal half-life (T1/2); area under the curve (AUC); maximum serum concentration (C max); time to reach Cmax (Tmax); volume of distribution (Vd); body clearance rate (CL) 6
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 7
Author Contributions
6
QD, JL, YY, and WH participated in research design. QD, XY, SD, and HY conducted experiments. QD and YZ performed data analyses. QD, JL, YY, and WH wrote or contributed to writing the manuscript. 8
Acknowledgments
This study was supported by the Science and Technology Commission of Shanghai Municipality (Grant 075407071). 9
References
Bardal, S. K., Waechter, J. E., and Martin DS. (2011). “Pharmacokinetics,” in Applied Pharmacology (St. Louis, MO: Saunders), 17-34. Kadomatsu, K., Kishida, S., and Tsubota, S. (2013). The heparin-binding growth factor midkine: the biological activities and candidate receptors. J. Biochem. 153, 511-521. Little, C. B., and Zaki, S. (2012). What constitutes an animal model of osteoarthritis–the need for consensus? Osteoarthr. Cartil. 20, 261-267. Matsuda, Y., and Talukder, A. H. (1996). Limited proteolysis by chymotrypsin of midkine and inhibition by heparin binding. Biochem. Biophys. Res. Commun. 228, 176-181. Muramatsu, T. (2014). Structure and function of midkine as the basis of its pharmacological effects. Br. J. Pharmacol. 171, 814-826. Noriyuki Suzuki, Yoshihisa Shibata, Takeshi Urano, Toyoaki Murohara, Takashi Muramatsu, Kenji Kadomatsu. (2004). Proteasomal Degradation of the Nuclear Targeting Growth Factor Midkine. The Journal of Biological Chemistry.279 (17): 17785–17791. Persky, A. M. (2013). Foundations in Pharmacokinetics. Chapel Hill, NC: UNC Press. EBOOK ISBN: 978-1-4696-3600-9. pp. 47-80. Qing Deng, Xiaolan Yu, Shaorong Deng, Hao Ye, Yang Zhang, Wei Han, Jingjing Li, Yan Yu. (2020). Midkine promotes articular chondrocyte proliferation through the MK LRP1-nucleolin signaling pathway. Cellular Signalling. 65,109423. Tandel, H., Florence, K., and Misra, A. (2011). “Protein and peptide delivery through respiratory pathway,” in Challenges in Delivery of Therapeutic Genomics and Proteomics, ed. A. Misra (London: Elsevier), 429-479. Yu, S. P., and Hunter D. J. (2016). Intra-articular therapies for osteoarthritis. Expert Opin. Pharmacother. 23-41. Zhang, Z. H., Li, H. X., Qi, Y. P., Du, L. J., Zhu, S. Y., Wu, M. Y., et al. (2010). Recombinant human midkine stimulates proliferation of articular chondrocytes. Cell Prolif. 43, 184-194. Zhou, H., and Theil, H. P. (2015). ADME and Translational Pharmacokinetics/Pharmacodynamics of Therapeutic Proteins: Applications in Drug Discovery and Development. Hoboken, NJ: John Wiley and Sons. ISBN: 978-1-118-89874-1. 7
Figure legends
Figure 1. Serum concentration-time curves after 1 mg/kg intramuscular injection of three types of rhMK in male SD rats, administered at time 0 h. Mean values ± SD are given, n = 6.
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Figure 2. Comparison of main pharmacokinetic parameters for three kinds of rhMK in rats after a single intramuscular injection. (A) T1/2; (B) AUC; (C) Cmax; (D) Tmax; (E) Vd/f; (F) CL/f. Mean values ± SD are given, n = 6. NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001 by a twotailed unpaired t-test.
9
Figure 3. Serum concentration-time curves after 1 mg/kg intramuscular or intraarticular injection of rhMK in male SD rats, administered at time 0 h. Mean values ± SD are given, n = 6.
Figure 4. Comparison of main pharmacokinetic parameters of rhMK in rats after a single intramuscular or intraarticular injection. (A) T1/2; (B) AUC; (C) Cmax; (D) Tmax; (E) Vd/f; (F) CL/f. 10
Mean values ± SD are given, n = 6. NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001 by a two-tailed unpaired t-test.
Figure 5. Serum rhMK concentration-time curve (A) and articular cartilage rhMK concentration-time curve (B) after 1 mg/kg intraarticular dose of rhMK to Hartley guinea pigs, administered at time 0 min. Mean values ± SD are given, n = 6.
Figure 6. Serum rhMK concentration-time curves after 10 mg/kg (A) or 50 mg/kg (B) intramuscular dose of rhMK to male SD rats, administered at time 0 min. Mean values ± SD are given, n = 6.
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Figure 7. Comparison of main pharmacokinetic parameters of rhMK in rats after a single intramuscular injection at a dose of 10 mg/kg or 50 mg/kg. (A) T1/2; (B) AUC; (C) Cmax; (D) Tmax; (E) Vd/f; (F) CL/f. Mean values ± SD are given, n = 6. NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001 by a two-tailed unpaired t-test.
Graphical abstract rhMK without extended N-terminal sequences expressed by Pichia displayed the best PK behavior in rats after single IM injection and exhibited non-linear PK behavior at dosages between 10 mg/kg and 50 mg/kg.
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Pharmacokinetics of Midkine with Different N-terminal Structures in Rats Qing Deng , Xiaolan Yu , Shaorong Deng , Hao Ye , Yang Zhang , Jingjing Li Ph.D. , Wei Han , Yan Yu Ph.D. PII: DOI: Reference:
S0928-0987(20)30090-7 https://doi.org/10.1016/j.ejps.2020.105301 PHASCI 105301
To appear in:
European Journal of Pharmaceutical Sciences
Received date: Revised date: Accepted date:
18 September 2019 9 January 2020 8 March 2020
Please cite this article as: Qing Deng , Xiaolan Yu , Shaorong Deng , Hao Ye , Yang Zhang , Jingjing Li Ph.D. , Wei Han , Yan Yu Ph.D. , Pharmacokinetics of Midkine with Different Nterminal Structures in Rats, European Journal of Pharmaceutical Sciences (2020), doi: https://doi.org/10.1016/j.ejps.2020.105301
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier B.V.
Pharmacokinetics of Midkine with Different N-terminal Structures in Rats Qing Deng1,2, Xiaolan Yu1, Shaorong Deng2, Hao Ye2, Yang Zhang2, Jingjing Li2, Wei Han2, Yan Yu1. 1
Shanghai Municipality Key Laboratory of Veterinary Biotechnology, School of Agriculture and Biology, Shanghai Jiao Tong University, NO.800, Dongchuan Road, Shanghai, 200240, China. 2 Laboratory of Regeneromics, School of Pharmacy, Shanghai Jiao Tong University, NO.800, Dongchuan Road, Shanghai, 200240, China. * Correspondence: Yan Yu, Ph.D. Tel.: +86-021-34204902; Fax: +86-021-34204902; E-mail address: [email protected]. Jingjing Li, Ph.D. Tel.: +86-021-34205760; Fax: +86-021-34205760; E-mail address: [email protected]. Abstract Midkine (MK) is a heparin-binding growth factor that functions in multiple physiological processes, making it a promising drug target for treating various diseases including osteoarthritis (OA). However, the lack of pharmacokinetic studies on MK limits further clinical research. As the Ndomain of MK protein appears to be more important for its stability, this study aimed to investigate the pharmacokinetic profiles of recombinant human (rh)MK with different structures at the Nterminus via different administration routes in rats and guinea pigs. A single intramuscular (IM) injection of 1 mg/kg rhMK with or without extended sequences at the N-terminus expressed by Escherichia coli or Pichia was administered to six male SD rats. rhMK concentrations in sequential tail blood samples were measured by ELISA. rhMK without extended N-terminal sequences expressed by Pichia had a greater area under the curve (AUC), slower clearance, and longer half-life in rats following a single IM injection than those of the other rhMK proteins. The AUC values for rhMK after IM and intra-articular (IA) administration were 1523.3 ± 35.2 h×ng/mL and 872.0 ± 36.1 h×ng/mL, whereas the apparent volumes of distribution (Vd/f) were 0.184 ± 0.067 L/kg and 11.6 ± 0.8 L/kg, respectively, suggesting that rhMK was distributed more locally after IA injection than after IM injection as Vd/f magnitude gives a general idea of extent distribution in the body and higher Vd/f represents more locally distribution. rhMK concentration in the articular cartilage was markedly higher than that in serum and reached the highest level at 3 days after a single IA injection in Hartley guinea pigs. As the dose increased from 10 to 50 mg/kg, the AUC increased in a greaterthan-dose-proportional manner, suggesting that rhMK exhibits non-linear pharmacokinetics in rats after a single IM injection in this dose range. These results indicated that the N-terminal structure and administration route have substantial effects on the pharmacokinetics of rhMK in rats. Furthermore, rhMK was maintained in articular cartilage with minimal diffusion into the blood following IA injection in Hartley guinea pigs, providing a foundation for clinical research on the use of rhMK for OA treatment via IA delivery. Keywords: midkine, pharmacokinetic, N-terminal, intramuscular, intraarticular
1
Introduction
Midkine (MK) is a heparin-binding growth factor; it belongs to a small protein family with pleiotrophin (Muramatsu, 2014). MK enhances the survival, migration, cytokine expression, differentiation, and other activities of target cells. MK has important effects on various physiological processes, such as development, reproduction, and repair. It is also involved in the pathogenesis of inflammatory and malignant diseases (Kadomatsu et al., 2013). With respect to its structure, MK includes two main domains: an N-terminal N-domain and a C-terminal C-domain. The two domains are relatively independent with no detectable interactions. The C-domain contributes to major MK functions including neurite-promoting activity. The N-domain appears to be more important for protein stability (Muramatsu, 2014). Furthermore, due to a difference in the cleavage of the signal sequence, there are two forms of MK, differing in the presence of two extended amino acids (valinealanine or other amino acids) at the N-terminus. Thus, alteration of the N-terminal sequence is of great interest for the production of MK with a longer half-life (Muramatsu, 2014). Different routes of administration may be used to achieve either systemic or local protein and peptide delivery. For small therapeutic molecules, the drug delivery routes include parenteral (intravenous, intramuscular, and subcutaneous), oral, nasal, ocular, transmucosal (buccal, vaginal, and rectal), and transdermal. However, the routes for the administration of proteins and peptides are limited owing to their large size and structure. Active ingredients must be delivered directly to the target site in a rapid, easy, and convenient manner (Tandel et al., 2011). IM administration of drugs in aqueous solution results in rapid absorption of the drug in most cases, and drug absorption is influenced by factors that alter blood flow to the muscle (Bardal et al., 2011). We previously demonstrated that MK has a significant repair effect in rat models of articular cartilage injury and promotes the proliferation of articular chondrocytes in vitro and in vivo through IM injection (Zhang et al., 2010). Therefore, MK might serve as a candidate drug for osteoarthritis (OA). Several drugs are used to treat OA via intraarticular (IA) administration (Yu and Hunter, 2016); thus, IA injection of MK is a potential therapeutic approach for OA. Pharmacokinetic (PK) studies are of great importance to pave the way for clinical research on MK. In this study, we evaluated the pharmacokinetics of an extended N-terminal sequence expressed in Escherichia coli or without the extended N-terminal sequence expressed in both E. coli or Pichia, and determined the rhMK protein with ideal pharmacokinetic behavior in rats for further research. We also compared the PK parameters for different administration routes. These results provide a basis for further clinical studies on MK for OA treatment. 2
Materials and methods
2.1
Animals
Six-week-old male Sprague–Dawley (SD) rats and male 6-month-old Hartley guinea pigs were used. All rats and guinea pigs were housed in a barrier facility with 2 ppm chlorinated water, autoclaved food, and bedding, under a 12-h light–dark cycle and 40% humidity. Animals were provided free access to food and water. The animals were fasted for 12 h prior to dosing and for the duration of the experiment. The experimental protocols were approved by the Animal Care and Use Committee of School of Pharmacy, Shanghai Jiao Tong University (Animal Ethic Approval Number#2017052). 2
2.2
Reagents
The sources of the materials used were as follows: Recombinant Human Midkine (rhMK) (Jiaochen General Regeneratives (Shanghai) Limited, Shanghai, China); Human Midkine DuoSet ELISA (Cat#332410, R&D, Minneapolis, MN, USA); BCA Assay Kit (Beyotime, China). 2.3
Study design
All rats or Hartley guinea pigs were weighed (six SD rats or six Hartley guinea pigs per group). At 0 min, 1 mg/kg or 10 mg/kg rhMK was given IM or IA. 100 μL blood samples were then obtained from each rat by tail blood sampling at 5 min, 10 min, 30 min, 1 h, 2 h, 4 h, 6 h, 8 h, 12 h, and 24 h post-injection. The blood was collected in tubes and centrifuged for 30 min at 3000 rpm to collect the serum. The serum samples were stored at –80°C until analysis. 2.4
Total protein extraction from articular cartilage
Cartilage from the femoral condyles of knee joints in Hartley guinea pigs was diced and stored in liquid nitrogen after a single IA injection of rhMK (1 mg/kg) at 0 min, 1 h, 1 d, 3 d, 6 d, 9 d, 12 d. Total protein from frozen tissue was extracted using Bead Ruptor 24 Elite (OMNI, USA) according to manufacturer’s instructions. After extraction, the processed tissue was kept on ice for 30 min and centrifuged for 15 min (14,000 × g, 4°C). The concentration of the extracted protein was measured using the BCA Assay Kit and the rhMK concentration in articular cartilage was measured using the Human Midkine DuoSet ELISA Kit. rhMK concentration was normalized to the total protein concentration from articular cartilage to obtain the relative rhMK concentration in articular cartilage. 2.5
Enzyme-linked immunosorbent assay (ELISA)
rhMK concentrations in blood samples were analyzed by Human Midkine DuoSet ELISA according to the manufacturer’s instructions. Briefly, 100 μL of standard or 10-fold diluted serum sample by blank rat serum was incubated in a 96-well microplate (pre-coated with goat anti-human MK antibody) for 2 h at room temperature (25°C). Following three washes, biotinylated goat anti-human MK antibody was added and incubated with captured MK for 2 h at room temperature (25°C). After three further washes, 100 μL of streptavidin-conjugated horseradish-peroxidase was added and allowed to react for 30 min in the dark. The plate was then washed and the substrate solution (1:1 mixture of H2O2 and tetramethylbenzidine) was added to the wells (100 μL per well) for a 20-min reaction. Finally, 1 mol/L H2SO4 (stop solution) was added (50 μL per well) and the absorbance of the wells was measured at 450 nm. The sample concentrations were then determined after generating a standard curve using a four-parameter logistic curve-fit method. 2.6
Statistical analysis
A two-compartment model was selected to obtain the PK parameters of rhMK in rats using WinNonlin3.3. AUC, Cmax, Tmax and k (the elimination constant rate) were calculated using the above software and following were computation methods of T1/2, CL/f and Vd/f: T1/2=0.693/k, CL/f=dose/AUC, Vd/f=CL/f×T1/2/0.693. Results are expressed as means ± standard deviation (SD). Statistically significant differences among treatment groups were determined using a two-tailed unpaired t-test in Graphpad. P < 0.05 indicated significant differences. 3
Results
3.1
PK profiles for three kinds of rhMK protein in rats after single intramuscular injection 3
To investigate the effects of an extended N-terminal region of MK on PK progression, we analyzed three kinds of rhMK protein in rats after a single IM injection. The total blood volume of rat weighed 200-250 g was 11 mL-17.5 mL according to the Guidance for Nonclinical Pharmacokinetics of Medicinal Products” issued by National Medical Products Administration (NMPA). We collected 100 μL blood sample from each rat at pointed time and the total collected volume during 24 h was 1 mL (less than 10% of total blood volume).The serum concentration-time curves for rhMK with or without extended N-terminal sequences expressed by E. coli or Pichia after single IM injection are shown in Figure 1. The results are shown in Figure 2 and Supplemental Table 1. The maximum serum concentrations (Cmax) of rhMK without the extended N-terminal sequences expressed by E. coli (E.coli rhMK (KKK)) (503.0 ± 67.7 ng/mL) and Pichia (Pichia rhMK (KKK)) (465.6.0 ± 12.9 ng/mL) were much greater than those of rhMK with extended N-terminal sequences expressed by E. coli (E.coli rhMK (VAKKK)) (198.1 ± 87.7 ng/mL). The Tmax for E. coli rhMK (KKK) and Pichia rhMK (KKK) was 0.9 ± 0.2 h and 1.0 ± 0.6 h, respectively, which was shorter than that for E.coli rhMK (VAKKK) (1.8 ± 0.5 h). The area under the curve (AUC) for Pichia rhMK (KKK) (1523.3 ± 35.2 h×ng/mL) was significantly higher than that of E. coli rhMK (KKK) (1278.4 ± 110.3 h×ng/mL, p = 0.0059) and E. coli rhMK (VAKKK) (861.8 ± 36.8h×ng/mL, p < 0.0001). However, the terminal half-lives (T1/2) of E. coli rhMK (VAKKK), E. coli rhMK (KKK), and Pichia rhMK (KKK) were 1.4 ± 0.4 h, 2.5 ± 1.1 h, and 2.4 ± 0.8 h, respectively, and did not differ significantly. The volume of distribution (Vd/f) and body clearance rate (CL/f) of Pichia rhMK (KKK) (0.184 ± 0.067 L/kg; 0.0537 ± 0.002 L/h/kg) were lowest among those for the three kinds of rhMK. These results support the use of Pichia rhMK (KKK) in subsequent research. 3.2
Comparison of PK behavior of rhMK following intramuscular or intraarticular administration in rats
The serum concentration-time curves for Pichia rhMK (KKK) via IM injection and IA injection are shown in Figure 3. The Cmax of Pichia rhMK (KKK) following IM delivery (465.6 ± 12.9 ng/mL) was significantly higher than that after IA injection (303.7 ± 33.3 ng/mL, p < 0.0001). The AUC and Tmax values for Pichia rhMK (KKK) during IM injection (1523.3 ± 35.2 h×ng/mL; 1.0 ± 0.6 h) were markedly higher than those for IA injection (872.0 ± 36.1 h×ng/mL, p < 0.0001 ; 0.5 ± 0.0 h, p = 0.0005), whereas the T1/2 values for Pichia rhMK (KKK) after administration by IM or IA routes were similar at 2.4 ± 0.8 h and 1.9 ± 0.1 h, respectively. However, the Vd/f and CL/f values for Pichia rhMK (KKK) after IM injection (0.184 ± 0.067 L/kg; 0.0537 ± 0.002 L/h/kg) were considerably lower than those of Pichia rhMK (KKK) after IA injection (11.6 ± 0.8 L/kg, p < 0.0001; 4.2 ± 0.2 L/h/kg, p < 0.0001) (Figure 4, Supplemental Table 2). 3.3
Pichia rhMK (KKK) storage in articular cartilage of Hartley guinea pigs after intraarticular injection
The serum rhMK concentration-time curve and articular cartilage rhMK concentration-time curve after IA injection are shown in Figure 5. The maximum rhMK serum concentration was detected at 10 min after dosing, followed by a rapid decrease. The articular cartilage rhMK concentration reached the highest level at 3 days after IA injection and a relatively high concentration was maintained for about 9 days. 3.4
Effect of dosage on rhMK PK profiles in rats
Serum PK parameters for Pichia rhMK (KKK) were evaluated after IM administration using doses of 10 mg/kg or 50 mg/kg, as summarized in Figures 6–7 and Supplemental Table 3. For Pichia rhMK (KKK) following IM injection with different doses, the increases in AUC and C max were dose4
dependent (10 mg/kg: 1298.4 ± 199.3 h×ng/mL < 50 mg/kg: 10139.4 ± 497.7 h×ng/mL, p < 0.0001; 10 mg/kg: 524.6 ± 198.9 ng/mL < 50 mg/kg: 3399.4 ± 769.2 ng/mL, p = 0.0033). However, T1/2 and Tmax values for Pichia rhMK (KKK) at doses of 10 mg/kg or 50 mg/kg did not differ significantly after IM injection. CL/f was decreased as the dose increased, from 0.039 L/h/kg at 10 mg/kg to 0.005 L/h/kg at 50 mg/kg (p=0.007). Vd/f ranged from 0.124 to 0.017 L/kg at the tested doses (p = 0.0006). 4
Discussion
We performed a PK analysis of rhMK with or without extended N-terminal sequences expressed by E. coli or Pichia in rats after IM or IA injection. An isoform with two additional amino acids (valine-alanine or other amino acids) at the N-terminus is present in MK preparations from different species due to a difference in the cleavage of the signal sequence. The N domain appears to be more important for the stability of MK. Therefore, to produce MK with a longer half-life, the extended form that has an additional VA at the N terminus, is of great interest. Limited proteolysis of MK by chymotrypsin is specifically inhibited by binding to heparin (Matsuda and Talukder, 1996) and MK with the extended N-terminus binds heparin more weakly compared to MK without the extended sequence (Muramatsu, 2014). In this study, we compared the main PK parameters of rhMK with or without extended N-terminal sequences expressed by E. coli and rhMK without an extended N-terminal sequence expressed by Pichia (Figures 1–2). The similar Cmax, T1/2, and Tmax values of E.coli rhMK(KKK) and Pichia rhMK(KKK) suggested that the different protein expression systems did not influence the rate of rhMK absorption in rats after a single IM injection. However, considerable differences in PK parameters between E. coli rhMK (KKK) and E. coli rhMK (VAKKK) suggested that the extended sequence at the N terminus regulated the absorption, distribution, and clearance of rhMK in rats after a single IM injection. rhMK without the extended N terminal sequence showed better absorption, as evidenced by the higher AUC and Cmax and lower Tmax. This kind of rhMK protein was distributed mainly in the circulation and displayed slower elimination, as indicated by lower Vd/f and CL/f values. Therefore, we used the rhMK protein without the extended N terminal sequence as our target for subsequent analyses. In a comparison of the main PK parameters for E. coli rhMK (KKK) and Pichia rhMK (KKK), we found that rhMK expressed by Pichia displayed better absorption and slower elimination, with a higher AUC and lower CL/f. Based on these results, rhMK without the extended N terminal sequence expressed by Pichia was found ideal for further pharmacokinetic research. Different administration routes affect PK progression for the same protein under identical experimental conditions (Persky, 2013). We previously found that rhMK is involved in promoting proliferation of rat chondrocytes in vivo and in vitro through IM injection (Zhang et al., 2010). Thus, we conducted a PK study of rhMK after a single IM or IA injection (Figures 3–4) and evaluated the articular cartilage rhMK concentration after IA delivery in rats (Supplemental Figure 4). The data suggested that less rhMK enters the circulation is due to a more local distribution after IA injection than after IM injection, as indicated by the lower AUC and C max and higher Vd/f as well as the storage of relative high concentration of rhMK in articular cartilage for almost 9 days. However, the terminal half-life of rhMK after IM injection and IA injection did not differ markedly. Furthermore, Hartley guinea pigs are widely used experimental animals for preclinical studies of OA (Little and Zaki, 2012), and thus, we evaluated the serum rhMK concentration and the relative rhMK concentration in articular cartilage following single IA injection in Hartley guinea pigs (Figure 5). rhMK was poorly absorbed into circulation, as indicated by the low C max of rhMK in the serum after IA injection. MK reached the highest concentration 3 days after injection and was maintained in the
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articular cartilage for almost 9 days. These results illustrate that rhMK can be maintained in articular cartilage to exert its repair function. Some therapeutic protein drugs display non-linear PK behavior in humans (Zhou and Theil, 2015). To evaluate whether or not MK is one such protein drug, we performed a PK study of rhMK after IM injection with 10 mg/kg or 50 mg/kg (Figures 6–7). The results suggest that absorption increases with an increase in dosage after IM injection. However, we also found that the Vd/f and CL/f values for rhMK following IM injection decrease as the dose increases. This can potentially be explained by target-mediated drug disposition, which occurs when binding to the pharmacodynamic target structure and affects the PK dynamics of a drug compound, resulting in capacity-limited saturable processes. The consequence of these saturable processes, caused by the limited availability of enzymes, receptors, or other protein structures that interact with the drug, is non-linear PK behavior, including higher clearance and a larger apparent volume of distribution at lower doses (Zhou and Theil, 2015). Previously, we demonstrated that MK promotes the proliferation of chondrocytes by LRP1 mediated endocytosis (Deng, 2020), thus, LRP1 might be one of the targets interacting with MK to affect its PK behavior. In this study, we found that rhMK in rats after IM injection exhibits non-linear pharmacokinetic behavior; in particular, the dose-normalized concentration vs. time profiles at different dosages is non-superimposable for IM injection. Thus, the CL/f and Vd/f for rhMK were higher at lower doses following IM injection. Noriyuki Suzuki has demonstrated that lysosomal system and proteasomal system are involved in the degradation of endocytosed midkine in embryonic fibroblasts in vitro (Noriyuki, 2004). However, the elimination process of rhMK in rats or humans is still unclear and needs to be clarified. This is the first PK study of MK with different structures at the N-terminus administered through various injection routes. We obtained the following main results. 1) The extended form of the Nterminus of rhMK has a great effect on its pharmacokinetic characteristics and rhMK without extended N-terminal sequences expressed by Pichia displays the best PK behavior in rats after a single IM injection among the three rhMK proteins evaluated. 2) rhMK was maintained in articular cartilage and exhibited little diffusion into circulation after single IA administration in rats and guinea pigs. 3) rhMK exhibited non-linear PK behavior in rats after IM injection within a dose range of 10–50 mg/kg. The PK profiles of MK with different structures administered through different routes described in this study improve our understanding of the pharmacology of MK and provide a basis for its clinical use in mammals. 5
Abbreviations
Midkine (MK); intramuscular (IM); intraarticular (IA); terminal half-life (T1/2); area under the curve (AUC); maximum serum concentration (C max); time to reach Cmax (Tmax); volume of distribution (Vd); body clearance rate (CL) 6
Conflict of Interest
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. 7
Author Contributions
6
QD, JL, YY, and WH participated in research design. QD, XY, SD, and HY conducted experiments. QD and YZ performed data analyses. QD, JL, YY, and WH wrote or contributed to writing the manuscript. 8
Acknowledgments
This study was supported by the Science and Technology Commission of Shanghai Municipality (Grant 075407071). 9
References
Bardal, S. K., Waechter, J. E., and Martin DS. (2011). “Pharmacokinetics,” in Applied Pharmacology (St. Louis, MO: Saunders), 17-34. Kadomatsu, K., Kishida, S., and Tsubota, S. (2013). The heparin-binding growth factor midkine: the biological activities and candidate receptors. J. Biochem. 153, 511-521. Little, C. B., and Zaki, S. (2012). What constitutes an animal model of osteoarthritis–the need for consensus? Osteoarthr. Cartil. 20, 261-267. Matsuda, Y., and Talukder, A. H. (1996). Limited proteolysis by chymotrypsin of midkine and inhibition by heparin binding. Biochem. Biophys. Res. Commun. 228, 176-181. Muramatsu, T. (2014). Structure and function of midkine as the basis of its pharmacological effects. Br. J. Pharmacol. 171, 814-826. Noriyuki Suzuki, Yoshihisa Shibata, Takeshi Urano, Toyoaki Murohara, Takashi Muramatsu, Kenji Kadomatsu. (2004). Proteasomal Degradation of the Nuclear Targeting Growth Factor Midkine. The Journal of Biological Chemistry.279 (17): 17785–17791. Persky, A. M. (2013). Foundations in Pharmacokinetics. Chapel Hill, NC: UNC Press. EBOOK ISBN: 978-1-4696-3600-9. pp. 47-80. Qing Deng, Xiaolan Yu, Shaorong Deng, Hao Ye, Yang Zhang, Wei Han, Jingjing Li, Yan Yu. (2020). Midkine promotes articular chondrocyte proliferation through the MK LRP1-nucleolin signaling pathway. Cellular Signalling. 65,109423. Tandel, H., Florence, K., and Misra, A. (2011). “Protein and peptide delivery through respiratory pathway,” in Challenges in Delivery of Therapeutic Genomics and Proteomics, ed. A. Misra (London: Elsevier), 429-479. Yu, S. P., and Hunter D. J. (2016). Intra-articular therapies for osteoarthritis. Expert Opin. Pharmacother. 23-41. Zhang, Z. H., Li, H. X., Qi, Y. P., Du, L. J., Zhu, S. Y., Wu, M. Y., et al. (2010). Recombinant human midkine stimulates proliferation of articular chondrocytes. Cell Prolif. 43, 184-194. Zhou, H., and Theil, H. P. (2015). ADME and Translational Pharmacokinetics/Pharmacodynamics of Therapeutic Proteins: Applications in Drug Discovery and Development. Hoboken, NJ: John Wiley and Sons. ISBN: 978-1-118-89874-1. 7
Figure legends
Figure 1. Serum concentration-time curves after 1 mg/kg intramuscular injection of three types of rhMK in male SD rats, administered at time 0 h. Mean values ± SD are given, n = 6.
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Figure 2. Comparison of main pharmacokinetic parameters for three kinds of rhMK in rats after a single intramuscular injection. (A) T1/2; (B) AUC; (C) Cmax; (D) Tmax; (E) Vd/f; (F) CL/f. Mean values ± SD are given, n = 6. NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001 by a twotailed unpaired t-test.
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Figure 3. Serum concentration-time curves after 1 mg/kg intramuscular or intraarticular injection of rhMK in male SD rats, administered at time 0 h. Mean values ± SD are given, n = 6.
Figure 4. Comparison of main pharmacokinetic parameters of rhMK in rats after a single intramuscular or intraarticular injection. (A) T1/2; (B) AUC; (C) Cmax; (D) Tmax; (E) Vd/f; (F) CL/f. 10
Mean values ± SD are given, n = 6. NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001 by a two-tailed unpaired t-test.
Figure 5. Serum rhMK concentration-time curve (A) and articular cartilage rhMK concentration-time curve (B) after 1 mg/kg intraarticular dose of rhMK to Hartley guinea pigs, administered at time 0 min. Mean values ± SD are given, n = 6.
Figure 6. Serum rhMK concentration-time curves after 10 mg/kg (A) or 50 mg/kg (B) intramuscular dose of rhMK to male SD rats, administered at time 0 min. Mean values ± SD are given, n = 6.
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Figure 7. Comparison of main pharmacokinetic parameters of rhMK in rats after a single intramuscular injection at a dose of 10 mg/kg or 50 mg/kg. (A) T1/2; (B) AUC; (C) Cmax; (D) Tmax; (E) Vd/f; (F) CL/f. Mean values ± SD are given, n = 6. NS, not significant, *P < 0.05, **P < 0.01, ***P < 0.001 by a two-tailed unpaired t-test.
Graphical abstract rhMK without extended N-terminal sequences expressed by Pichia displayed the best PK behavior in rats after single IM injection and exhibited non-linear PK behavior at dosages between 10 mg/kg and 50 mg/kg.
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