Studies on Stability of Alprostadil (Lipo-PGE1 and DN-PGE1) Targeting Preparation

Fan Xu1,2*, Cheng Wu3 and Su Zeng1

1Institute of Drug Metabolism and Pharmacesutical Analysis, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, 310058, China

2920 Hospital of Joint Logistics Support Force, Kunming, 650032, China

3Department of Health Statistics, Naval Medical University, Shanghai, 200433, China

ABSTRACT

Objective of this study was to investigate the compatibility and stability of alprostadil (lipo-PGE1 and DN-PGE1) after compatibility with different volume of 0.9% sodium chloride or 5% glucose, and to provide a basis for drug safe use in the clinic. PGE1 lipid microspheres (lipo-PGE1) and alprostadil dried emulsion (DN-PGE1) were compatibility with different volumes of 0.9% sodium chloride and 5% glucose, respectively. After the compatibility of different concentrations, the changes of pH, particle size distributions, the particles greater than 5μm and encapsulation rate of the compatibility solutions were observed within 8 h by using electronic acidimeter, Malvern zeta sizer nanoparticle size analyzer, accusizer APS 780 particle size analyzer and Agilent 6460 LC-MS/MS mass spectrometer. From the experimental results, 5 μg:50ml group kept good targeting within 6 h. The encapsulation rate of 5 μg:100ml group decreased significantly after 6 h, and the percentage of fat globules > 5 μ m (PFAT5) in 10 μg:10ml group was relatively high. The risk of embolism was easily caused by injection or small pot dripping, which may be related to the insufficient amount of solvent and the incomplete dispersion of lipid microspheres. As for the two dosage forms of lipo-PGE1 and DN-PGE1, the average particle size and PFAT5 after the preparation of DN-PGE1met the USP standard, and the stability indexes were more advantageous. The clinical treatment of alprostadil targeted injection can be prepared by pharmacy intravenous admixture service. The drug concentration should be more than 0.05 μg/ml, and the drug concentration 0.1 μg/ml is the most appropriate. Lipo-PGE1 and DN-PGE1 injection can be administered by intravenous drip. The finished infusion should be used up within 6 h after the dispensing.

Article Information

Received 22 July 2020

Revised 28 August 2020

Accepted 16 September 2020

Available online 12 August 2021

(early access)

Published 24 April 2022

Authors’ Contribution

FX and SZ conceived the idea of the research and designed the experiments. FX conducted the experiments. FX and CW analyzed the results. All authors discussed the results and wrote the manuscript.

Key words

Alprostadil injections, Lipo-PGE1, DN-PGE1, Stability

DOI: https://dx.doi.org/10.17582/journal.pjz/20200722050704

* Corresponding author: Xu_fan@126.com

0030-9923/2022/0004-1927 $ 9.00/0

Copyright 2022 by the authors. Licensee Zoological Society of Pakistan.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Prostaglandin E1 (PGE1), another name as alprostadil, is an autacoid drug. It is effective in treating patients with various peripheral vascular occlusive disorders (Martin and Tooke, 1982) and with spontaneous pain and sensory disturbance due to diabetic neuropathy and diabetic ulcers (Low et al., 1986). However, the clinical application of PGE1 is limited due to its adverse reactions to frequently causes local pain on administration and low bioavailability. To overcome these problems, several new alternative dosage forms of PGE1, such as lipid microspheres (Mizushima et al., 1983), cyclodextrin clathrate (Yabek and Mann, 1979), nanoparticles (Ishihara et al., 2008) and nano emulsion (Preece, 2017) can prevent PGE1 from inactivation in blood and improve the efficacy of PGE1. Among these new dosage forms, cyclodextrin clathrate prostaglandin E1 (PGE1-CD) injected intravenously is rapidly inactivated in the lungs and a high dose is necessary for the treatment of these disorders furthermore renal toxicity of beta-cyclodextrin limited its clinical use (Lewis et al., 1981; Peled et al., 1992). PGE1 lipid microspheres (lipo-PGE1) which incorporated PGE1 into lipid microspheres were established by Mizushima et al. (1983) and alprostadil dried emulsion (DN-PGE1) which incorporated PGE1 into nanoemulsion and freeze-dried at low temperature by Chongqing Yaoyou Pharmaceutical Co., Ltd. Since they can deliver the encapsulated PGE1 efficiently to disease sites, lipo-PGE1 and DN-PGE1 were used widely in the clinic.

This type of alprostadil targeted formulation clearly states in its instructions: Alprostadil injection is administered once a day for adults, 1-2ml (5-10μg of alprostadil) + 10ml of normal saline (or 5% Glucose) intravenously (Okuno et al., 2020; Fan et al., 2019). In clinical application, how to formulate this type of infusion to ensure the safety and effectiveness of alprostadil is a matter of great concern. In this study, the stability of alprostadil targeted formulations (alprostadil lipid microsphere injection and alprostadil lipid microsphere dry emulsion) in different volumes and different kinds of solvent were investigated by using comparative experimental methods. We aimed to provide a basis for the safe use of this formulation in clinical practice.

Materials and methods

Alprostadil (99%) was purchased from Taiwan Yongguang Chemical Co., China; E0018506 and Alprostadil injection from Beijing Teide Pharmaceutical Co., China; 1B058H, 1B068H, 1B078H and Alprostadil emulsion from Chongqing Youyao Pharmaceutical Co., China; 18220960, 18220190, 18220020 were used for the experiment.

For measurement of precision and accuracy Alprostadil was dissolved in methanol and diluted to 20, 5, and 1 ng/ml and used for injection. Each concentration was measured six times and was injected on different days. Three batches were prepared and measured to calculate intra-batch and inter-batch precision and accuracy. The results showed that the intra-precisions among three concentrations (RSD) were 8.52%, 5.92%, and 7.12%. The inter-precisions among three concentrations (RSD) were 11.14%, 6.83%, and 9.52%. The accuracy of three concentrations (RE) was 12.14%, 6.83%, and -5.52%. The results show that the precision and accuracy of alprostadil is good under the conditions of this study.

For measurement of stability alprostadil was dissolved in methanol and diluted to 20 and 1 ng/ml. Each concentration was prepared for 6 samples. Three samples were placed in the sampler, and measured after being placed for 0 h, 2h, 4h, and 8h; Other three samples were placed at room temperature and measured after being placed for 0 h, 2 h, 4 h, and 8 h. The results showed that the stability of alprostadil was good.

For measurement of appearance and pH three new prostol preparations Kaishi, Mencito, and Youdier were thoroughly mixed with saline injection or 5% glucose injection, to prepare three concentrations of 1.0, 0.1, and 0.05 μg/ml. This operation was reported for 3 batches of each alprostadil targeted preparation product. The appearance of compatibility solution was observed and then pH was recorded using Mettler Seven Easy pH meter, Mettler Toledo Instruments, Switzerland.

Two ml samples of the compatibility solution of prostol preparations Kaishi and Youdier were taken at 0, 1, 2, 3, 4, 6, and 8h, after prepration and the changes in average particle size and 90% cumulative particle size were measured with the Malvern Zetasizernano particle size analyzer (Malvin Instruments, UK). Three parallel tests were conducted for each rinse solution. Particles greater than 5 μm were detected by Accusizer 780/ APS (PSS particle size analyzer company, USA). Three parallel tests were conducted for each punch.

For measurement of encapsulation rate at different times, 1ml of each rinse solution was sampled on the gel column (Hitrap gel column, 5ml), and then eluted with acetic acid buffer at pH 4.5 (see Chinese pharmacopeia buffer). 5 ml was dissolved by isopropanol into 25 ml. After shaking, the sample solution 1 was obtained, and the enveloped PGE1 was determined by LC-MS/MS (Agilent 6460 LC -MS/MS mass spectrometer, USA). Another 1 ml sample was dissolved by isopropanol into 25 ml. After shaking, the sample solution 2 was obtained, and the enveloped PGE1 was also determined by LC-MS/MS. The encapsulation rate is calculated according to the following formula:

Encapsulation rate %= encapsulation PGE1/ total PGE1×100

For determination of alprostadil and its degradation product PGA1 at different times,1 ml of each rinse solution was dissolved by isopropanol into 10 ml. After shaking, the sample solution was obtained. LC-MS/MS was used to determine alprostadil and PGA1 in the sample solution.

For LC-MS/MS chromatographic conditions column were Agilent XDB C18 (2.1 × 50mm, 1.8µm); mobile phase: A is 0.05% glacial acetic acid aqueous solution, B is 0.05% glacial acetic acid methanol solution; flow rate: 0.3ml / min; injection volume: 20µl; with gradient wash in LC-MS/MS as shown below.

Time (min): 0, 4, 4.01, 6; A% 35, 0, 35, 35. MS conditions ESI ion source; dry gas flow rate: 8 L/min, dry gas temperature: 350 oC, atomizing gas pressure: 25 psi, capillary voltage: -3500 V; negative ion detection. Scanning method is Multiple Reaction Monitoring (MRM). The ions are: parent ion 353.2, daughter ion 317.2, fragmentation voltage 110 V, and collision energy 16 V.

Results and discussion

Comparing the pH and average particle size difference of two different dosage forms of alprostadil at different diluent volumes in two different solvents, the results showed that the difference of solvent type and placement time was not statistically significant. There were differences in pH and average particle size between dosage forms, and the pH value of lipo-PGE1 was lower after punching and the mean particle size of the lipo-PGE1 was larger after punching. The influence of dilution volume on pH and the mean particle size were different. The pH of 5μg:100ml was lower and the mean particle size was greated than 10μg:10ml, and the difference between 5μg:50ml and 10μg:10ml was not statistically significant. There was no difference in dosage, solvent, and volume over time (Table I).

Comparing the PFAT5 and encapsulation rateschanges of two different dosage forms of alprostadil in two different solvents with different dilution volume, the results showed that the difference of dosage type, solvent type, dilution volume and placement time was not statistically significant. There was no difference in the type of solvent over time. Compared with DN-PGE1, there were more particles larger than 5μmin lipo-PGE1 with the extension of the placement time. 5μg:50ml had the least number of particles greater than 5 μm over time, followed by 5μg:100ml and 10μg:10ml (Table I).

Table I. Analysis results of pH mean particle size, particles greater than 5μm and encapsulation rate variation mixed linear model.

Note: SAS 9.4 is adopted to take time as a random effect; dose, solvent, volume as fixed effect.

The encapsulation rate of 5μg:100ml was significantly lower than that of 10μg:10ml in different diluted volumes, and the difference between 5μg:50ml and 10μg:10ml was not statistically significant (Table I).

The instructions limit the solvent to 10ml and require immediate use. This is mainly because the use of large amounts of solvent will destroy the structure of lipid microspheres in the targeted drugs of alprosidil injection, which will dissolve the drug into the solvent. On the one hand, it will increase the incidence of adverse drug reactions (phlebitis); on the other hand, it will also destroy the targeting of the drug. However, other clinical studies on the treatment of different diseases by intravenous infusion of 50-100ml 0.9% sodium chloride after infusion of proprodil targeting preparations and achieved good therapeutic effects (Liang et al., 2018; Hao et al., 2017; Wang et al., 2016). Tian et al. (2017) conducted a systematic evaluation of phlebitis caused by different administration methods of alprostadil injection. Intravenous infusion of alprostadil was more recommended by the conclusion.

However, from the perspective of infection control and infusion safety, the safety of intravenous drug dispensing centers for treatment infusion is much higher than that of nurses in an exposed environment. Intravenous injection not only increases the difficulty of clinical nursing but also increases the risk of infection. Therefore, the clinical application of alprostadil targeted injection by intravenous drip is relatively common, and the treatment is effective (Liang et al., 2018; Yi-na et al., 2014).

Since the inner diameter of the venous capillaries is 4~9 μm, in a clinical continuous infusion, excessive particles with a diameter of > 5 μm may block the capillaries after entering the systemic circulation, leading to the inflammatory response. Particle retention in the lungs will cause pulmonary embolism and liver function impairment (Yi-na et al., 2014). Therefore, the size and size distribution of lipo-particles are important indexes to evaluate the stability and safety of injection. The United States Pharmacopoeia (USP) (Kelley et al., 2012) stipulates that MDD < 500 nm and PFAT5≤ 0.05%. The national drug standard (Hutt, 2008) stipulates that MDD should be 120~280 nm. In this study, the dynamic light scattering method was used to measure MDD, which has a low sensitivity to the ions with a diameter of >1 μm. Therefore, light shading-single particle optical sensing technology (LO/SPOS) was used to detect PFAT5. LO/SPOS technology is highly sensitive to large liposomes. It can accurately measure the size and number of large liposomes by detecting and counting the large size particles one by one, instead of obtaining an approximate value based on mathematical transformation. Encapsulation rate is an important index to evaluate the preparation technology and quality of lipid microspheres, and it is also the key to improve drug treatment index, reduce adverse drug reactions, and reduce drug dose. In this study, gel chromatography was used to separate lipid microspheres from free drugs by the difference in molecular weight and particle size. The larger size of the lipid microspheres was eluted first, and the smaller size of the free drug was eluted to achieve the separation effect. After separation, the enriched samples were completely dissolved with 4 times the volume of isopropanol. The content of PGE1 was determined by LC-MS /MS and the encapsulation rate was calculated.

According to the experimental results, the 5μg: 50ml group maintained good targeting of the preparation within 6h. The encapsulation rate of 5 μg:100ml group decreased significantly after 6h. The percentage of PFAT5 in 10 μg:10ml group was higher. The risk of embolization is easily caused by push injection or small pot infusion, which may be related to the insufficient amount of solvent and the inability to completely disperse the lipid microspheres. As for lipo-PGE1 injection and DN-PGE1 injection, the average particle size and PFAT5 percentage of DN-PGE1 are in line with USP standards, and the stability indicators are more dominant.

Conclusion

The clinical treatment of alprostadil targeted injection can be dispensed by pharmacy intravenous admixture service. The concentration of the drug should over 0.05 μg /ml, and the concentration of the drug 0.1 μg /ml is the most appropriate. The drug can be administered by intravenous infusion, and the finished infusion should be used within 6 h after deployment.

Acknowldegments

The research is supported by the program of training young and middle-aged academic and technical leaders in Yunnan Province (No. 2017HB052).

Supplementary material

There is supplementary material associated with this article. Access the material online at: https://dx.doi.org/10.17582/journal.pjz/20200722050704

Statement of conflict of interest

The authors have declared no conflict of interest.

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