HM-3 is a new anti-tumor polypeptide developed in our laboratory, which is composed of 18 amino acids and previous studies showed that the target of HM-3 is integrin (αvβ3) [1]. In vivo and in vitro studies have shown that HM-3 could inhibit endothelial cell migration and angiogenesis, thereby inhibiting tumor growth. HM-3 is expected to be developed as therapeutic drugs for the treatment of solid tumors including gastric and liver cancer [2]. HM-3 has been authorized as a national invention patent and declared for an international patent [3].

However, similar to other peptide drugs, the half-life of HM-3 is short (approximately 25 min). HM-3 is easily degraded by proteases [1,4]. Animal studies suggested that the optimal therapeutic efficiency could be obtained by administration of the drug twice a day.

An effective approach to solve this problem is to modify peptide drugs with polymers such as polyethylene glycol (PEG) and dextran [5]. Currently, most studies are focusing on PEG modification [6–8]. PEG is a class of large polymers with unique physical and chemical properties. PEG is non-toxic, non-antigenic and has good biocompatibility [9,10]. The main biological function of PEG-modified protein or peptide drugs remains unchanged. More importantly, PEG modification increases the circulatory stability, extends the half-life, reduces the immunogenicity and decreases the toxcity of protein or peptide drugs [11–14].

However, PEG modification still exhibits several problems in application. Based on the literature [9,10], the main problems associated with PEG modification were an abnormal increase of molecular weight of the modified products and a decrease or loss of activity during the process of modification. Loss of activity is essentially the primary problem that needs to be solved for PEG modification of peptide drugs [15,16].

Selection of the appropriate molecular size of PEG is key for the modification of peptide drugs. Two main considerations for selecting the type of PEG are the residual in vivo biological activity and the renal elimination rate of the modified products. The molecular size of PEG is negatively related to the residual biological activity of the peptide drugs [17]. The relationship between the size of PEG and renal elimination rate is that a larger size of PEG normally results in a lower renal elimination rate. Studies have shown that the renal elimination rate when using PEG with a molecular weight of 20 kDa is approximately 10%, which plays a significant role in increasing the half-life, extending drug administration frequency and maintaining the blood drug concentration at a high level [18,19]. Currently, a variety of PEG-modified protein and peptide drugs have been developed and approved by the FDA for biomedical purposes [20].

ALD-mPEG and SC-mPEG are two different types of PEG, which are currently the most commonly used and relatively mature with active groups to modify polypeptide drugs. The active groups of ALD-mPEG and SC-mPEG are propionaldehyde and succinimidyl carbonate, respectively, which are specificity reactive groups of the peptide’s end amino. Physical properties, reaction activity with the peptide’s end amino of ALD-mPEG and SC-mPEG were different, and the remaining bioactivity of the same peptide PEGylated by the different two PEG were different too. A previous study [1] reported that HM-3 modified with ALD-mPEG_10k showed improved half-life and biological activity in vitro, but the half-life improvement was not significant.

In order to solve the problem of short half-life and rapid plasma clearance, our laboratory utilized four different types of PEG (ALD-mPEG and SC-mPEG) with different molecular weights to modify the N-terminus of HM-3. We expected that the modified products would have a long half-life while maintaining the anti-tumor activity. Our results showed that the modified product, SC-mPEG_20k-HM-3, had a low plasma clearance rate, long half-life and retained its anti-tumor activity excellently. These results provide a fundamental basis for the development of new anti-tumor drugs.

It is well known that attachment of polyethylene glycol (PEG) to therapeutic peptides or proteins (referred to as PEGylation) effectively prolongs their half-lives, reduces antigenicity and immunogenicity, and improves their pharmacokinetic and pharmacodynamic properties, rendering the drugs significantly prolonged circulating half-lives [23,24].

SC-mPEG (mPEG-succinimidyl carbonate) and ALD-mPEG (mPEG-propionaldehyde) have been the most commonly used chain-shaped PEG with active groups. Urethane bonds generated after coupling between SC-mPEG and peptides, and secondary amine generated after combining ALD-mPEG and peptides, are more stable and have a higher hydrolytic stability than ester bonds [25,26]. The SC-mPEG was extremely reactive, with reactions with peptides or proteins requiring higher pH in PBS, while the ALD-mPEG required mild reaction conditions, and could combine with peptides or proteins in mild pH in PBS [27]. Yet, the reactivity of the latter was lower than the former using the same peptides or proteins.

In PEGylation of pharmaceuticals, the design of an appropriate conjugation strategy is essential to confer the desired properties to the parent peptide or protein. The random conjugation of PEG would result in significant decrease, even loss of bioactivities, which showed inconsistent therapeutic effects and was undesired in PEGylated pharmaceuticals [20,28,29].

Therefore, when we modify a molecule with PEG, we should choose PEGs that have optimal balance between the activity maintaining rate and their half-life in vivo. According to previous studies, the in vitro activity of the PEGylated HM-3 products (especially SC-mPEG_20k-HM-3) were high, but the in vivo activity was not measured. Thus, it is necessary to study the in vivo activity of the PEGylated HM-3 [1,4].

Figure 6 shows that the activity maintaining rate of modification by SC-mPEG_10k and ALD-mPEG_10k were not high, and the tumor inhibition rate of SC-mPEG_10k-HM-3 (20.27%) was lower than ALD-mPEG_10k-HM-3 (31.64%), while the modified product of SC-mPEG_20k-HM-3 (50.23%) was excellent, displaying similar tumor inhibitory activity in vivo to the Taxol and unmodified HM-3 groups.

Generally speaking, modification with large molecule PEG may result in reduced activity. The activity of modified products of anti-tumor peptides and proteins is closely associated with organism resistance, PEG mass and shape, stability of PEG-peptide or PEG-protein bond, and changes in the spatial structure of modified products [27,30].

In our study, the activity of SC-mPEG_20k-HM-3 did not change. This might be due to the fact that the loss of biological activity was compensated for by the significantly prolonged half-life and blood plasma residence time in vivo, as a result of the increased stability and higher hydrodynamic volume after modification. The activity of ALD-mPEG_5k-HM-3, ALD-mPEG_10k-HM-3, and SC-mPEG_10k-HM-3 would decline with different degrees after modification, while the half-life and blood plasma residence time of these in vivo were low, which were not obviously prolonged in the previous study. Perhaps these factors cause the anti-tumor activity of ALD-mPEG_5k-HM-3, ALD-mPEG_10k-HM-3, and SC-mPEG_10k-HM-3 to be lower than SC-mPEG_20k-HM-3 and unmodified HM-3 in vivo. Besides, the active groups of SC-mPEG and ALD-mPEG are different, coupling between SC-mPEG and peptides with urethane bond, and in the presence of sodium cyanoborohydride, a secondary amine would be generated after combination of ALD-mPEG and peptides. The latter stability was higher than the former in vivo [31,32]. Maybe this led to the different activity of SC-mPEG_10k-HM-3 and ALD-mPEG_10k-HM-3 in vivo.

SC-mPEG_20k-HM-3 retained the original biological activity of HM-3. Tumor inhibition of SC-mPEG_20k-HM-3 (50.23%) was slightly higher than that of Taxol in vivo. These data suggest that the half-life of HM-3 can be improved by modification with SC-mPEG_20k. For instance, drug administration frequency was extended from twice per day to once per two days after modification by SC-mPEG_20k. The anti-tumor therapeutic efficacy of SC-mPEG_20k-HM-3 was more efficient than that of HM-3. The body weight of mice in each group was maintained at a stable level, indicating that the effective dosage of drugs is non-toxic or the toxicity is low.

Therefore, this study demonstrated that SC-mPEG_20k can be used for modification of HM-3 and further determined in vivo biological activity of the modified product SC-mPEG_20k-HM-3. We will further characterize the physio-chemical properties, in vivo pharmacokinetics, immunogenicity and the antitumor target of SC-mPEG_20k-HM-3.

Our preliminary results found that although the tumor grew slowly and the tumor formation rate was low in nude mice inoculated with 5 × 10⁵ tumor cells/mice, administration of drugs had a good effect in inhibiting the tumors. The tumor inhibition for small tumors (20 mm³–30 mm³) was more efficient than for large tumors (>80 mm³). Taxol administered once per two days (10 mg/kg/time) had better tumor inhibition than that administered twice per week. However, Taxol is highly toxic and frequent administration of taxol may result in weight loss or even death. Thus, in this study, we treated the mice with Taxol twice per week. In addition, if the tumor cell line was transferred in culture too many times, the property of the cells may be changed, which may affect the evaluation of the therapeutic efficiency. These results provide references for other related studies.

SC-mPEG_20k-HM-3 retained the anti-tumor activity of HM-3 better than other PEG-modified products. In addition, SC-mPEG_20k modified peptide HM-3 had a low plasma clearance rate, long half-life and retained its anti-tumor activity. Therefore, SC-mPEG_20k-HM-3 has a very high value for development of new anti-tumor drugs.