http://informahealthcare.com/ddi ISSN: 0363-9045 (print), 1520-5762 (electronic) Drug Dev Ind Pharm, Early Online: 1–7 ! 2015 Informa Healthcare USA, Inc. DOI: 10.3109/03639045.2015.1040417
RESEARCH ARTICLE
Viscoelastic interactions between polydeoxyribonucleotide and ophthalmic excipients
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Iksoo Kim, Hyeongmin Kim, Kyunghee Park, Sandeep Karki, Prakash Khadka, Kanghee Jo, Seong Yeon Kim, Jieun Ro, and Jaehwi Lee Pharmaceutical Formulation Design Laboratory, College of Pharmacy, Chung-Ang University, Seoul, Korea
Abstract
Keywords
This study investigated the interaction between polydeoxyribonucleotide (PDRN) and several ionic and nonionic isotonic agents, thickeners and a preservative that were employed as excipients in ophthalmic preparations. Interaction of each individual excipient and PDRN aqueous solution was evaluated by analyzing their rheological properties. Rheological properties of PDRN solutions were evaluated by dynamic oscillatory shear tests and values of elastic modulus (G0 ), viscous modulus (G00 ) and loss tangent (tan ) were used to assess the relative changes in viscoelastic properties. At given concentrations, sodium chloride was found to show alteration in viscoelastic properties of PDRN solution while nonionic isotonic agents like D-glucose and D-sorbitol did not alter them. Similarly, nonionic water soluble polymers like polyvinylpyrrolidone (PVP) and hydroxypropyl methylcellulose (HPMC) also did not interact with PDRN to alter the viscoelastic properties. However, there were changes observed when carbopol 940 was used as a thickener. Therefore, PDRN was found to interact with ionic excipients and the interactions were negligible when nonionic materials were examined, which suggests that nonionic excipients are suitable to be formulated with PDRN.
Polydeoxyribonucleotide, ophthalmic formulation, pharmaceutical excipients, rheology, viscoelasticity
Introduction Polydeoxyribonucleotide (PDRN) is a biopolymer consisting of randomly arranged deoxyribonucleotides with chain lengths ranging from 50 to 2000 base pairs. It is generally obtained from sperm and testicles of trout or salmon with a selective extraction process to attain over 95% purity. PDRN is considered to be a source of purines and pyrimidines when delivered to the body, thereby stimulating nucleic acid synthesis through the salvage pathway1. It is also known to bind with adenosine A2A receptors, a subclass of purinergic receptors, resulting in the reduction of inflammation, and the stimulation of angiogenesis, collagen synthesis and the proliferation of a variety of cells such as osteoblasts, fibroblasts, pre-adipocytes and endothelial cells2–4. Owing to these properties, PDRN has been increasingly used as a bio-revitalizing agent for the regeneration of mucosa, bone, cartilage and tendon5–7. Additionally, numerous clinical researches have demonstrated substantial would-healing properties of PDRN 2,8–10. Furthermore, PDRN is regarded as a safe and biocompatible drug when considering its constituents1,3,11. In clinical practice, PDRN is also considered to be a promising ophthalmic therapeutic agent for the corneal epithelial wound
Address for correspondence: Prof. Jaehwi Lee, Pharmaceutical Formulation Design Laboratory, College of Pharmacy, Chung-Ang University, Seoul 156-756, South Korea. Tel: +82-2-820-5606. Fax: +82-2-816-7338. E-mail: [email protected]
History Received 13 January 2015 Revised 20 March 2015 Accepted 9 April 2015 Published online 29 May 2015
because, in addition to the tissue regenerative activities described above, it can stimulate the healing process of the corneal epithelium12 and can be used as a source of metabolic energy for the corneal tissues to maintain their physiological functions13,14. It is thus necessary to develop PDRN ophthalmic formulations, and as a dosage form, eye drops are thought to be desirable due to the clinically proven advantages and preference by the patients15. However, fastidious attention is necessary when designing ophthalmic formulations of PDRN because it might be expected that PDRN interacts with frequently used ophthalmic excipients such as isotonic agents and thickeners. PDRN presents numerous negative charges that stem from phosphodiester backbones in its chemical structure and thus, the positively charged excipients are able to bind with PDRN, probably leading to the changes in solution properties including changes in the rheological characteristics. For instance, polyvinylpyrrolidone (PVP), alkylated cellulose derivatives and polyethylene glycol that are typically employed as thickeners in ophthalmic formulations, have caused DNA condensation affecting viscoelastic properties of the solution16–18. Therefore, the aim of this study was to investigate the interactions occurring between PDRN and ophthalmic excipients by analyzing the rheological properties of ophthalmic PDRN solutions. Ophthalmic excipients such as isotonic agents (sodium hydrochloride, D-glucose and D-sorbitol)19, thickeners [PVP K30, PVP K90, hydroxypropyl methylcellulose (HPMC) 2910, and sodium polyacrylate (Carbopol) 940]20,21, and a preservative (benzalkonium chloride)22,23 were selected for this study based on
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their common use in ophthalmic formulations. The impact of these ophthalmic excipients on rheological properties of PDRN solutions was evaluated with a dynamic oscillatory shear test.
Materials and methods Materials
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PDRN was provided by Pharma Research Products Co., Ltd. (Seongnam, Korea). Sodium chloride and D-sorbitol were obtained from Daejung Chemicals & Metals Co., Ltd. (Siheung, Korea) and D-glucose was purchased from Samchun Chemicals (Yeosu, Korea). PVP K30 and K90 were purchased from BASF (Cleveland, OH). HPMC 2910 was supplied by Whawon Pharm Co., Ltd. (Seoul, Korea). Carbopol 940 was provided by Lubrizol Advanced Materials (Cleveland, OH). Benzalkonium chloride was purchased from Sigma-Aldrich Company (St. Louis, MO) and all other chemicals used were of pharmaceutical grade. Preparation of PDRN ophthalmic formulations As shown in Table 1, PDRN formulations containing various ophthalmic excipients were prepared by adding one milliliter of excipient solutions to nine milliliter of PDRN solution (PDRN dissolved in 9 ml double distilled water) and the formulations were subsequently vortex mixed for 3 min. Evaluation of rheological characteristics Dynamic oscillatory shear tests were conducted to evaluate rheological behaviors of PDRN formulations using a rotational rheometer (HAAKE RheostressÔ 1, Thermo Fisher Scientific Inc., Waltham, MA) fitted with a 2 6 cm titanium cone (C60/2 Ti L, Thermo Fisher Scientific Inc.). The gap between the cone and plate was set at 1.04 mm. Each test sample (3–4 mL) was carefully loaded with a micropipette on the rheometer plate maintained at 20 ± 0.1 C and allowed to equilibrate for 5 min prior to the measurements. Under the measuring conditions employed, the test samples were securely resided within the cone and plate geometry. Initially, torque sweeps were carried out to find torque ranges demonstrating linear viscoelastic characteristics at an oscillation frequency of 1 rad/s. The oscillatory shear tests were then performed over oscillatory frequency ranges of 0.1–80 rad/s.
Rheological data analysis Two rheological parameters G0 (elastic modulus) and G00 (viscous modulus) were measured with HAAKE RheoWin software (Ver. 4.30.0016, Thermo Fisher Scientific Inc.) as a function of frequency concurrently. The values of loss tangent (tan ) were obtained by the relationship of G00 /G0 in order to assess relative changes in viscoelastic properties of PDRN formulations24. Osmolality measurements Under the influence of the ophthalmic excipients, the osmolality of PDRN formulations was measured to confirm if it was in the tolerable range of tonicity. Osmolality of each formulation was measured using a freezing point osmometer (Osmomat 030; Gonotec GmbH, Berlin, Germany). Before measuring the osmolality of PDRN formulations, distilled water (50 ml) was placed in a measuring tube, and its osmolality was measured to set a zero point of osmolality. Calibration was also performed with a calibration standard solution of which osmolality was 300 mOsm/kg (Calibration Standard; Gonotec GmbH, Berlin, Germany). Statistical analysis All measurements were performed in triplicate. Rheological data indicate mean values and standard deviation bars are omitted for clarity. The relative standard deviations were 510% in all measured values. Osmolality values are represented as mean ± standard deviation.
Results and discussion The present study investigated the effect of the ophthalmic excipients on the bulk rheological properties of aqueous PDRN solution to design efficient ophthalmic formulations of PDRN. The interaction of ophthalmic formulations with the tear film would be of critical importance for the maintenance of normal physiology of the eye and drug efficacy25,26. Therefore, the evaluation of the surface rheology of the tear film in the presence of ophthalmic formulations might be performed but due to lack of proper equipment this was not part of the current study. Effect of isotonic agents on viscoelastic property It was necessary to adjust the tonicity of PDRN ophthalmic formulations to be within the tolerable tonicity range for the eye
Table 1. Composition of PDRN formulations. The volume of all formulations was adjusted to 10 ml. Sample No. F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F16 F17 F18 F19 F20
PDRN (mg)
Isotonic agent
Thickener
Preservative
Amount of ophthalmic exipient added (mg)
300 300
– Sodium chloride
– –
– –
300
D-glucose
–
–
300
D-sorbitol
–
–
300
–
PVP K30
–
300
–
PVP K90
–
300 300 300
– – –
HPMC 2910 Carbopol 940 –
– – Benzalkonium chloride
– 18.75 37.5 75 125 250 500 125 250 500 50 150 450 1350 150 450 1350 50 50 1.0
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[169–508 mOsm/kg corresponding to the osmolality of 0.5–1.5% (w/v) sodium chloride solution] because an extremely hypertonic or hypotonic ophthalmic solution may cause pain and other undesirable symptoms when applied27,28. Isotonic agents are therefore generally added to ophthalmic formulations to control their tonicity. In this study, three different isotonic agents were added to PDRN formulations, and subsequent changes in the viscoelastic characteristics were analyzed to investigate interactions between isotonic agents and PDRN. Sodium chloride, D-glucose and D-sorbitol were chosen as model isotonic agents as they have widely been used to control the tonicity of ophthalmic formulations due to their good water solubility and biocompatibility29–31. Figure 1 shows the profiles of changes in G0 , G00 and tan measured from a PDRN formulation devoid of any ophthalmic excipients (F1) and PDRN formulations containing sodium chloride (F4), D-glucose (F7) and D-sorbitol (F10) as a function of the oscillation frequency between 0.1 and 80 rad/s. For F1, a
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dominant elastic response was seen with a plateau region where G0 exhibited an increasing trend as a function of the frequency, followed by stabilization to a plateau. The reason for this might be largely because entanglements among PDRN that can enhance the viscoelastic properties could be formed by the oscillatory shear stress applied at the PDRN concentration tested32. In terms of tan , they were lower than one and exhibited a characteristic curve at the whole frequency range, also implying that F1 showed predominant elastic property. PDRN formulations containing non-ionic isotonic agents such as D-glucose and D-sorbitol (F7 and F10) showed considerably similar viscoelastic behavior to F1 at the whole oscillation frequencies examined. This result demonstrates that the non-ionic isotonic agents did not largely interact with PDRN enough to significantly change the viscoelastic properties of the PDRN formulations. In contrast to F1, F7 and F10, the PDRN formulation incorporating sodium chloride (F4) showed significantly changed viscoelastic characteristics under the oscillatory shearing. G0 increased from 315.10 to 423.90 Pa as a function of the oscillation frequency, which values were considerably larger than those measured from F1, F7 and F10. In addition, the plateau of G0 were seen at lower oscillation frequencies than in the case of F1, F7 and F10. G00 were also greater than those measured from F1, F7 and F10, but they exhibited a decreasing trend as a function of the oscillatory frequency while those measured from F1, F7 and F10 showed an increasing trend. Tan measured from F4 were lower than those determined from F1, F7 and F10 at the whole frequency, implying that F4 had stronger elastic property than F1, F7 and F10. Tan values of F4 also consistently decreased as a function of the oscillatory frequency whereas those analyzed from F1, F7 and F10 showed the characteristic curve. This result might be attributed to more compactly entangled PDRN in F4 compared to in F1, F7 and F10. When sodium chloride was added to the PDRN formulation, sodium ions were dissociated and might have interacted with negative charges of PDRN molecules to subsequently make them more compactly entangled, which could render the PDRN formulation more elastic under the oscillatory shearing. Effect of concentration of isotonic agents on viscoelastic property
Figure 1. Effect of isotonic agents on G0 (A), G00 (B) and tan (C) measured from PDRN formulations over the frequency range of 0.1–80 rad/s.
To further clarify the effect of the isotonic agents and their concentration on the viscoelastic properties of PDRN formulations, the dynamic oscillatory shear tests were performed with varying the concentration of the isotonic agents. Figure 2 illustrates the profiles of changes in G0 , G00 and tan measured from the formulations containing the three different isotonic agents at different concentrations (F2–F10). In the case of the PDRN formulation containing sodium chloride (F2–F4), it was found that G0 and G00 were considerably increased with increasing concentration of sodium chloride. The plateau region of G0 was also observed from low oscillation frequencies in all of PDRN formulations containing sodium chloride. Tan generally decreased with increasing concentration of sodium chloride. As for the solutions containing the non-ionic isotonic agents, D-glucose and D-sorbitol at three different concentrations (F5–F10), no significant changes in the viscoelastic moduli were observed at the whole frequency range compared to the PDRN formulation without any ophthalmic excipients (F1). Thus, as stated in ‘‘Effect of isotonic agents on viscoelastic property’’ section, similar interaction between PDRN and the ionic and non-ionic isotonic agents could be observed. This interaction is in accordance with the theory that nucleic acids tend to interact with metallic cations. As reported by Hackl and Blagoi33, DNA molecules in aqueous or mixed solutions can
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Figure 2. Effect of changing concentration of isotonic agents on G0 (A, D and G), G00 (B, E and H) and tan (C, F and I) measured from PDRN formulations over the frequency range of 0.1–80 rad/s.
interact with calcium ions rendering the DNA to be transformed into more compact form. Although DNA condensation and aggregation usually occurs with multivalent cations, the addition of sodium chloride has been reported to aggregate mononucleosomal DNA34. Zinchenko and Yoshikawa35 have also reported the higher potential of sodium ions (to that of potassium ions) to cause DNA compaction while a few computer simulated studies have also suggested that sodium ions can potentially bind to the phosphate group of nucleic acids36,37. Thus, the concentrationdependent increase in elastic moduli values observed from F2–F4 might be due to the increased binding of sodium ions with PDRN, causing aggregation and compaction. Effect of thickeners on viscoelastic property In the preparation of ophthalmic formulations, thickeners that are usually polymeric compounds are frequently added to increase the viscosity. The main benefit of adding thickeners is to increase the ocular contact time, thereby reducing the drainage rate and subsequently increasing drug bioavailability. However, in the case of PDRN ophthalmic formulations, interactions between PDRN and thickeners can occur and significantly affect the entire physical properties of the formulations. Therefore, we prepared PDRN formulations containing three different thickeners such as PVP K30 (F11), HPMC 2910 (F18) and carbopol 940 (F19), and investigated the interaction between PDRN and the thickeners by analyzing the viscoelastic properties. As shown in Figure 3, PDRN formulations containing PVP K30 (F11) and HPMC 2910 (F18) showed a similar rheological behavior to that of F1 at the entire oscillation frequency range, implying there was no significant interaction between PDRN and
the thickeners. In contrast, the PDRN formulation containing carbopol 940 (F19) exhibited significantly changed viscoelastic properties compared to F1. G0 and G00 measured from F19 increased as a function of oscillation frequency, but G0 were considerably lower than those of F1, thereby showing comparatively higher tan than those measured from F1. Thus, F19 demonstrated weaker elastic property than that of F1. This result could be due to the possible interaction between carbopol 940 and PDRN. Carbopol 940 in aqueous solution has been reported to undergo ionization to give ionized carboxyl groups38 so that it is supposed that the electronic repulsion between PDRN and carbopol 940 could occur, leading to decrease in entanglements of PDRN and subsequent attenuated elastic property of F19 under the applied shearing. Effect of concentration and molecular weight of PVP on viscoelastic property To further investigate the influence of concentration and molecular weight of thickeners on viscoelastic properties of PDRN formulations, PVP K30 and PVP K90 having average molecular weights of 40 000 and 360 000, respectively, were selected as model thickeners due to their good water solubility and no foaming property. In this examination, three different concentrations of PVP K30 and PVP K90 were examined (15, 45 and 135 mg/ml, F12–F17). Figure 4 illustrates profiles of changes in the rheological parameters measured from F12–F17 at the oscillation frequency range examined. In general, G0 , G00 and tan increased as the concentration and molecular weight of the thickener increased. Particularly, the PDRN formulation containing PVP K90 with the highest concentration exhibited considerably
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DOI: 10.3109/03639045.2015.1040417
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Figure 4. Effect of changing molecular weight and concentration of PVP on G0 (A), G00 (B) and tan (C) measured from PDRN formulations over the frequency range of 0.1–80 rad/s. Figure 3. Effect of thickeners on G0 (A) G00 (B) and tan (C) measured from PDRN formulations over the frequency range of 0.1–80 rad/s.
increased G0 and G00 showing no plateau region while the other PDRN formulations presented the plateau. The reason for the enhancement of the elastic property of PDRN formulations might be due to the effect of the concentration and molecular weight of thickeners on their entanglements and relaxation time. It has been known that increase in molecular weight and concentration of polymers in polymeric solutions can increase the entanglements and relaxation time of polymers in response to applied shear stresses39. The increased entanglements and relaxation time can subsequently cause enhanced elastic property of the polymeric solutions under the oscillatory shear stress applied39. Thus, it is necessary to add thickeners with suitable concentration and molecular weight to PDRN ophthalmic formulations although the non-ionic thickeners might not interact largely with PDRN. Effects of benzalkonium chloride on viscoelastic property When benzalkonium chloride was added to PDRN solutions as a preservative, there were no significant changes in the
viscoelastic properties compared to those assessed with F1 (Figure 5). Since benzalkonium chloride is a quaternary cationic compound, there is possibility of positively charged ions to interact with PDRN in aqueous solution. However, the results suggested that there was no or minimal viscoelastic interaction of benzalkonium chloride with PDRN which might be due to a very low concentration (0.01 g/dl) of benzalkonium chloride used in the solution. Benzalkonium chloride is reported to interact with DNA in a concentration dependent manner40, which might be the reason behind negligible effects on viscoelastic property of PDRN, at low concentration of benzalkonium chloride. Effect of ophthalmic excipients on osmotic pressure As shown in Figure 6, the osmolality of PDRN formulations containing isotonic agents and thickeners was measured to determine if it was in the tolerable range of tonicity for delivery to the eyes. In the case of the PDRN formulations containing the isotonic agents (F2–F10), osmolality was increased as a function of concentration of the isotonic agents. Osmolality measured from F4, F7 and F10 was within
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the tolerable range of tonicity where most people do not feel any pain and discomfort27,28. In the case of the PDRN formulations containing only thickeners (F11–F17), the osmolality was below the required tolerable range but there was a proportional increase in the osmolality with respect to the amount of polymers in the formulations as shown in Figure 6. There was no significant difference in osmolality values of samples containing equivalent amounts of either PVP K30 or PVP K90 as thickeners (F12, F13 and F14 compared with F15, F16 and F17, respectively). PVP K30, PVP K90, HPMC 2910 and carbopol 940 showed roughly similar values of osmolality at a given concentration. Water soluble polymers can be used in pharmaceutical formulations to adjust or maintain osmotic pressure. Organic polymers are widely used as osmotic agents in various dosage forms to design osmotically controlled delivery systems such as osmotic pumps, osmotic controlled release tablets41. Therefore nonionic polymers such as PVP and HPMC, that do not interact with PDRN or affect its viscoelastic properties significantly, are suitable to be used in ophthalmic preparations at concentrations that can maintain tonicity at a tolerable range. In our study, polymers were used as thickeners rather than isotonic agents. However, due to their minimal interaction with PDRN, PVP and HPMC can also be used as osmotic agents.
Conclusion
Figure 5. Effect of benzalkonium chloride on G0 (A), G00 (B) and tan (C) measured from PDRN formulations over the frequency range of 0.1–80 rad/s.
Figure 6. Osmolality measured from PDRN formulations. Asterisk represents tolerable tonicity range for the eye illustrated in this figure was referred to from a research literature published22.
Only in the case sodium chloride used as an isotonic agent, properties of sample were turned into packed gel and great changes in G0 and G00 were observed. Therefore it is reasonable to use D-glucose or D-sorbitol, instead of sodium chloride, as isotonic agents for ophthalmic PDRN formulations. Among the thickeners, PVP was found to be an ideal thickener because carbopol 940 was found to interact with PDRN and induced molecular structure changes while HPMC 2910 was unsuitable because foaming was observed when dissolved in water. Among PVP K30 and PVP K90, PVP K90 showed more increase in viscoelastic moduli of PDRN formulations while both had similar influence on osmotic pressure of the formulations when used at similar amounts. Thus, PVP K90 can be more favorable to be used as a thickener, considering its effects on the viscoelastic properties of PDRN solution. Benzalkonium chloride (at concentrations 50.1% w/v) can also be included into the ophthalmic formulations as an anti-bacterial preservative.
DOI: 10.3109/03639045.2015.1040417
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This research was supported by the Chung-Ang University Research Scholarship Grants in 2014.
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RESEARCH ARTICLE
Viscoelastic interactions between polydeoxyribonucleotide and ophthalmic excipients
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Iksoo Kim, Hyeongmin Kim, Kyunghee Park, Sandeep Karki, Prakash Khadka, Kanghee Jo, Seong Yeon Kim, Jieun Ro, and Jaehwi Lee Pharmaceutical Formulation Design Laboratory, College of Pharmacy, Chung-Ang University, Seoul, Korea
Abstract
Keywords
This study investigated the interaction between polydeoxyribonucleotide (PDRN) and several ionic and nonionic isotonic agents, thickeners and a preservative that were employed as excipients in ophthalmic preparations. Interaction of each individual excipient and PDRN aqueous solution was evaluated by analyzing their rheological properties. Rheological properties of PDRN solutions were evaluated by dynamic oscillatory shear tests and values of elastic modulus (G0 ), viscous modulus (G00 ) and loss tangent (tan ) were used to assess the relative changes in viscoelastic properties. At given concentrations, sodium chloride was found to show alteration in viscoelastic properties of PDRN solution while nonionic isotonic agents like D-glucose and D-sorbitol did not alter them. Similarly, nonionic water soluble polymers like polyvinylpyrrolidone (PVP) and hydroxypropyl methylcellulose (HPMC) also did not interact with PDRN to alter the viscoelastic properties. However, there were changes observed when carbopol 940 was used as a thickener. Therefore, PDRN was found to interact with ionic excipients and the interactions were negligible when nonionic materials were examined, which suggests that nonionic excipients are suitable to be formulated with PDRN.
Polydeoxyribonucleotide, ophthalmic formulation, pharmaceutical excipients, rheology, viscoelasticity
Introduction Polydeoxyribonucleotide (PDRN) is a biopolymer consisting of randomly arranged deoxyribonucleotides with chain lengths ranging from 50 to 2000 base pairs. It is generally obtained from sperm and testicles of trout or salmon with a selective extraction process to attain over 95% purity. PDRN is considered to be a source of purines and pyrimidines when delivered to the body, thereby stimulating nucleic acid synthesis through the salvage pathway1. It is also known to bind with adenosine A2A receptors, a subclass of purinergic receptors, resulting in the reduction of inflammation, and the stimulation of angiogenesis, collagen synthesis and the proliferation of a variety of cells such as osteoblasts, fibroblasts, pre-adipocytes and endothelial cells2–4. Owing to these properties, PDRN has been increasingly used as a bio-revitalizing agent for the regeneration of mucosa, bone, cartilage and tendon5–7. Additionally, numerous clinical researches have demonstrated substantial would-healing properties of PDRN 2,8–10. Furthermore, PDRN is regarded as a safe and biocompatible drug when considering its constituents1,3,11. In clinical practice, PDRN is also considered to be a promising ophthalmic therapeutic agent for the corneal epithelial wound
Address for correspondence: Prof. Jaehwi Lee, Pharmaceutical Formulation Design Laboratory, College of Pharmacy, Chung-Ang University, Seoul 156-756, South Korea. Tel: +82-2-820-5606. Fax: +82-2-816-7338. E-mail: [email protected]
History Received 13 January 2015 Revised 20 March 2015 Accepted 9 April 2015 Published online 29 May 2015
because, in addition to the tissue regenerative activities described above, it can stimulate the healing process of the corneal epithelium12 and can be used as a source of metabolic energy for the corneal tissues to maintain their physiological functions13,14. It is thus necessary to develop PDRN ophthalmic formulations, and as a dosage form, eye drops are thought to be desirable due to the clinically proven advantages and preference by the patients15. However, fastidious attention is necessary when designing ophthalmic formulations of PDRN because it might be expected that PDRN interacts with frequently used ophthalmic excipients such as isotonic agents and thickeners. PDRN presents numerous negative charges that stem from phosphodiester backbones in its chemical structure and thus, the positively charged excipients are able to bind with PDRN, probably leading to the changes in solution properties including changes in the rheological characteristics. For instance, polyvinylpyrrolidone (PVP), alkylated cellulose derivatives and polyethylene glycol that are typically employed as thickeners in ophthalmic formulations, have caused DNA condensation affecting viscoelastic properties of the solution16–18. Therefore, the aim of this study was to investigate the interactions occurring between PDRN and ophthalmic excipients by analyzing the rheological properties of ophthalmic PDRN solutions. Ophthalmic excipients such as isotonic agents (sodium hydrochloride, D-glucose and D-sorbitol)19, thickeners [PVP K30, PVP K90, hydroxypropyl methylcellulose (HPMC) 2910, and sodium polyacrylate (Carbopol) 940]20,21, and a preservative (benzalkonium chloride)22,23 were selected for this study based on
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their common use in ophthalmic formulations. The impact of these ophthalmic excipients on rheological properties of PDRN solutions was evaluated with a dynamic oscillatory shear test.
Materials and methods Materials
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PDRN was provided by Pharma Research Products Co., Ltd. (Seongnam, Korea). Sodium chloride and D-sorbitol were obtained from Daejung Chemicals & Metals Co., Ltd. (Siheung, Korea) and D-glucose was purchased from Samchun Chemicals (Yeosu, Korea). PVP K30 and K90 were purchased from BASF (Cleveland, OH). HPMC 2910 was supplied by Whawon Pharm Co., Ltd. (Seoul, Korea). Carbopol 940 was provided by Lubrizol Advanced Materials (Cleveland, OH). Benzalkonium chloride was purchased from Sigma-Aldrich Company (St. Louis, MO) and all other chemicals used were of pharmaceutical grade. Preparation of PDRN ophthalmic formulations As shown in Table 1, PDRN formulations containing various ophthalmic excipients were prepared by adding one milliliter of excipient solutions to nine milliliter of PDRN solution (PDRN dissolved in 9 ml double distilled water) and the formulations were subsequently vortex mixed for 3 min. Evaluation of rheological characteristics Dynamic oscillatory shear tests were conducted to evaluate rheological behaviors of PDRN formulations using a rotational rheometer (HAAKE RheostressÔ 1, Thermo Fisher Scientific Inc., Waltham, MA) fitted with a 2 6 cm titanium cone (C60/2 Ti L, Thermo Fisher Scientific Inc.). The gap between the cone and plate was set at 1.04 mm. Each test sample (3–4 mL) was carefully loaded with a micropipette on the rheometer plate maintained at 20 ± 0.1 C and allowed to equilibrate for 5 min prior to the measurements. Under the measuring conditions employed, the test samples were securely resided within the cone and plate geometry. Initially, torque sweeps were carried out to find torque ranges demonstrating linear viscoelastic characteristics at an oscillation frequency of 1 rad/s. The oscillatory shear tests were then performed over oscillatory frequency ranges of 0.1–80 rad/s.
Rheological data analysis Two rheological parameters G0 (elastic modulus) and G00 (viscous modulus) were measured with HAAKE RheoWin software (Ver. 4.30.0016, Thermo Fisher Scientific Inc.) as a function of frequency concurrently. The values of loss tangent (tan ) were obtained by the relationship of G00 /G0 in order to assess relative changes in viscoelastic properties of PDRN formulations24. Osmolality measurements Under the influence of the ophthalmic excipients, the osmolality of PDRN formulations was measured to confirm if it was in the tolerable range of tonicity. Osmolality of each formulation was measured using a freezing point osmometer (Osmomat 030; Gonotec GmbH, Berlin, Germany). Before measuring the osmolality of PDRN formulations, distilled water (50 ml) was placed in a measuring tube, and its osmolality was measured to set a zero point of osmolality. Calibration was also performed with a calibration standard solution of which osmolality was 300 mOsm/kg (Calibration Standard; Gonotec GmbH, Berlin, Germany). Statistical analysis All measurements were performed in triplicate. Rheological data indicate mean values and standard deviation bars are omitted for clarity. The relative standard deviations were 510% in all measured values. Osmolality values are represented as mean ± standard deviation.
Results and discussion The present study investigated the effect of the ophthalmic excipients on the bulk rheological properties of aqueous PDRN solution to design efficient ophthalmic formulations of PDRN. The interaction of ophthalmic formulations with the tear film would be of critical importance for the maintenance of normal physiology of the eye and drug efficacy25,26. Therefore, the evaluation of the surface rheology of the tear film in the presence of ophthalmic formulations might be performed but due to lack of proper equipment this was not part of the current study. Effect of isotonic agents on viscoelastic property It was necessary to adjust the tonicity of PDRN ophthalmic formulations to be within the tolerable tonicity range for the eye
Table 1. Composition of PDRN formulations. The volume of all formulations was adjusted to 10 ml. Sample No. F1 F2 F3 F4 F5 F6 F7 F8 F9 F10 F11 F12 F13 F14 F15 F16 F17 F18 F19 F20
PDRN (mg)
Isotonic agent
Thickener
Preservative
Amount of ophthalmic exipient added (mg)
300 300
– Sodium chloride
– –
– –
300
D-glucose
–
–
300
D-sorbitol
–
–
300
–
PVP K30
–
300
–
PVP K90
–
300 300 300
– – –
HPMC 2910 Carbopol 940 –
– – Benzalkonium chloride
– 18.75 37.5 75 125 250 500 125 250 500 50 150 450 1350 150 450 1350 50 50 1.0
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[169–508 mOsm/kg corresponding to the osmolality of 0.5–1.5% (w/v) sodium chloride solution] because an extremely hypertonic or hypotonic ophthalmic solution may cause pain and other undesirable symptoms when applied27,28. Isotonic agents are therefore generally added to ophthalmic formulations to control their tonicity. In this study, three different isotonic agents were added to PDRN formulations, and subsequent changes in the viscoelastic characteristics were analyzed to investigate interactions between isotonic agents and PDRN. Sodium chloride, D-glucose and D-sorbitol were chosen as model isotonic agents as they have widely been used to control the tonicity of ophthalmic formulations due to their good water solubility and biocompatibility29–31. Figure 1 shows the profiles of changes in G0 , G00 and tan measured from a PDRN formulation devoid of any ophthalmic excipients (F1) and PDRN formulations containing sodium chloride (F4), D-glucose (F7) and D-sorbitol (F10) as a function of the oscillation frequency between 0.1 and 80 rad/s. For F1, a
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dominant elastic response was seen with a plateau region where G0 exhibited an increasing trend as a function of the frequency, followed by stabilization to a plateau. The reason for this might be largely because entanglements among PDRN that can enhance the viscoelastic properties could be formed by the oscillatory shear stress applied at the PDRN concentration tested32. In terms of tan , they were lower than one and exhibited a characteristic curve at the whole frequency range, also implying that F1 showed predominant elastic property. PDRN formulations containing non-ionic isotonic agents such as D-glucose and D-sorbitol (F7 and F10) showed considerably similar viscoelastic behavior to F1 at the whole oscillation frequencies examined. This result demonstrates that the non-ionic isotonic agents did not largely interact with PDRN enough to significantly change the viscoelastic properties of the PDRN formulations. In contrast to F1, F7 and F10, the PDRN formulation incorporating sodium chloride (F4) showed significantly changed viscoelastic characteristics under the oscillatory shearing. G0 increased from 315.10 to 423.90 Pa as a function of the oscillation frequency, which values were considerably larger than those measured from F1, F7 and F10. In addition, the plateau of G0 were seen at lower oscillation frequencies than in the case of F1, F7 and F10. G00 were also greater than those measured from F1, F7 and F10, but they exhibited a decreasing trend as a function of the oscillatory frequency while those measured from F1, F7 and F10 showed an increasing trend. Tan measured from F4 were lower than those determined from F1, F7 and F10 at the whole frequency, implying that F4 had stronger elastic property than F1, F7 and F10. Tan values of F4 also consistently decreased as a function of the oscillatory frequency whereas those analyzed from F1, F7 and F10 showed the characteristic curve. This result might be attributed to more compactly entangled PDRN in F4 compared to in F1, F7 and F10. When sodium chloride was added to the PDRN formulation, sodium ions were dissociated and might have interacted with negative charges of PDRN molecules to subsequently make them more compactly entangled, which could render the PDRN formulation more elastic under the oscillatory shearing. Effect of concentration of isotonic agents on viscoelastic property
Figure 1. Effect of isotonic agents on G0 (A), G00 (B) and tan (C) measured from PDRN formulations over the frequency range of 0.1–80 rad/s.
To further clarify the effect of the isotonic agents and their concentration on the viscoelastic properties of PDRN formulations, the dynamic oscillatory shear tests were performed with varying the concentration of the isotonic agents. Figure 2 illustrates the profiles of changes in G0 , G00 and tan measured from the formulations containing the three different isotonic agents at different concentrations (F2–F10). In the case of the PDRN formulation containing sodium chloride (F2–F4), it was found that G0 and G00 were considerably increased with increasing concentration of sodium chloride. The plateau region of G0 was also observed from low oscillation frequencies in all of PDRN formulations containing sodium chloride. Tan generally decreased with increasing concentration of sodium chloride. As for the solutions containing the non-ionic isotonic agents, D-glucose and D-sorbitol at three different concentrations (F5–F10), no significant changes in the viscoelastic moduli were observed at the whole frequency range compared to the PDRN formulation without any ophthalmic excipients (F1). Thus, as stated in ‘‘Effect of isotonic agents on viscoelastic property’’ section, similar interaction between PDRN and the ionic and non-ionic isotonic agents could be observed. This interaction is in accordance with the theory that nucleic acids tend to interact with metallic cations. As reported by Hackl and Blagoi33, DNA molecules in aqueous or mixed solutions can
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Figure 2. Effect of changing concentration of isotonic agents on G0 (A, D and G), G00 (B, E and H) and tan (C, F and I) measured from PDRN formulations over the frequency range of 0.1–80 rad/s.
interact with calcium ions rendering the DNA to be transformed into more compact form. Although DNA condensation and aggregation usually occurs with multivalent cations, the addition of sodium chloride has been reported to aggregate mononucleosomal DNA34. Zinchenko and Yoshikawa35 have also reported the higher potential of sodium ions (to that of potassium ions) to cause DNA compaction while a few computer simulated studies have also suggested that sodium ions can potentially bind to the phosphate group of nucleic acids36,37. Thus, the concentrationdependent increase in elastic moduli values observed from F2–F4 might be due to the increased binding of sodium ions with PDRN, causing aggregation and compaction. Effect of thickeners on viscoelastic property In the preparation of ophthalmic formulations, thickeners that are usually polymeric compounds are frequently added to increase the viscosity. The main benefit of adding thickeners is to increase the ocular contact time, thereby reducing the drainage rate and subsequently increasing drug bioavailability. However, in the case of PDRN ophthalmic formulations, interactions between PDRN and thickeners can occur and significantly affect the entire physical properties of the formulations. Therefore, we prepared PDRN formulations containing three different thickeners such as PVP K30 (F11), HPMC 2910 (F18) and carbopol 940 (F19), and investigated the interaction between PDRN and the thickeners by analyzing the viscoelastic properties. As shown in Figure 3, PDRN formulations containing PVP K30 (F11) and HPMC 2910 (F18) showed a similar rheological behavior to that of F1 at the entire oscillation frequency range, implying there was no significant interaction between PDRN and
the thickeners. In contrast, the PDRN formulation containing carbopol 940 (F19) exhibited significantly changed viscoelastic properties compared to F1. G0 and G00 measured from F19 increased as a function of oscillation frequency, but G0 were considerably lower than those of F1, thereby showing comparatively higher tan than those measured from F1. Thus, F19 demonstrated weaker elastic property than that of F1. This result could be due to the possible interaction between carbopol 940 and PDRN. Carbopol 940 in aqueous solution has been reported to undergo ionization to give ionized carboxyl groups38 so that it is supposed that the electronic repulsion between PDRN and carbopol 940 could occur, leading to decrease in entanglements of PDRN and subsequent attenuated elastic property of F19 under the applied shearing. Effect of concentration and molecular weight of PVP on viscoelastic property To further investigate the influence of concentration and molecular weight of thickeners on viscoelastic properties of PDRN formulations, PVP K30 and PVP K90 having average molecular weights of 40 000 and 360 000, respectively, were selected as model thickeners due to their good water solubility and no foaming property. In this examination, three different concentrations of PVP K30 and PVP K90 were examined (15, 45 and 135 mg/ml, F12–F17). Figure 4 illustrates profiles of changes in the rheological parameters measured from F12–F17 at the oscillation frequency range examined. In general, G0 , G00 and tan increased as the concentration and molecular weight of the thickener increased. Particularly, the PDRN formulation containing PVP K90 with the highest concentration exhibited considerably
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Figure 4. Effect of changing molecular weight and concentration of PVP on G0 (A), G00 (B) and tan (C) measured from PDRN formulations over the frequency range of 0.1–80 rad/s. Figure 3. Effect of thickeners on G0 (A) G00 (B) and tan (C) measured from PDRN formulations over the frequency range of 0.1–80 rad/s.
increased G0 and G00 showing no plateau region while the other PDRN formulations presented the plateau. The reason for the enhancement of the elastic property of PDRN formulations might be due to the effect of the concentration and molecular weight of thickeners on their entanglements and relaxation time. It has been known that increase in molecular weight and concentration of polymers in polymeric solutions can increase the entanglements and relaxation time of polymers in response to applied shear stresses39. The increased entanglements and relaxation time can subsequently cause enhanced elastic property of the polymeric solutions under the oscillatory shear stress applied39. Thus, it is necessary to add thickeners with suitable concentration and molecular weight to PDRN ophthalmic formulations although the non-ionic thickeners might not interact largely with PDRN. Effects of benzalkonium chloride on viscoelastic property When benzalkonium chloride was added to PDRN solutions as a preservative, there were no significant changes in the
viscoelastic properties compared to those assessed with F1 (Figure 5). Since benzalkonium chloride is a quaternary cationic compound, there is possibility of positively charged ions to interact with PDRN in aqueous solution. However, the results suggested that there was no or minimal viscoelastic interaction of benzalkonium chloride with PDRN which might be due to a very low concentration (0.01 g/dl) of benzalkonium chloride used in the solution. Benzalkonium chloride is reported to interact with DNA in a concentration dependent manner40, which might be the reason behind negligible effects on viscoelastic property of PDRN, at low concentration of benzalkonium chloride. Effect of ophthalmic excipients on osmotic pressure As shown in Figure 6, the osmolality of PDRN formulations containing isotonic agents and thickeners was measured to determine if it was in the tolerable range of tonicity for delivery to the eyes. In the case of the PDRN formulations containing the isotonic agents (F2–F10), osmolality was increased as a function of concentration of the isotonic agents. Osmolality measured from F4, F7 and F10 was within
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the tolerable range of tonicity where most people do not feel any pain and discomfort27,28. In the case of the PDRN formulations containing only thickeners (F11–F17), the osmolality was below the required tolerable range but there was a proportional increase in the osmolality with respect to the amount of polymers in the formulations as shown in Figure 6. There was no significant difference in osmolality values of samples containing equivalent amounts of either PVP K30 or PVP K90 as thickeners (F12, F13 and F14 compared with F15, F16 and F17, respectively). PVP K30, PVP K90, HPMC 2910 and carbopol 940 showed roughly similar values of osmolality at a given concentration. Water soluble polymers can be used in pharmaceutical formulations to adjust or maintain osmotic pressure. Organic polymers are widely used as osmotic agents in various dosage forms to design osmotically controlled delivery systems such as osmotic pumps, osmotic controlled release tablets41. Therefore nonionic polymers such as PVP and HPMC, that do not interact with PDRN or affect its viscoelastic properties significantly, are suitable to be used in ophthalmic preparations at concentrations that can maintain tonicity at a tolerable range. In our study, polymers were used as thickeners rather than isotonic agents. However, due to their minimal interaction with PDRN, PVP and HPMC can also be used as osmotic agents.
Conclusion
Figure 5. Effect of benzalkonium chloride on G0 (A), G00 (B) and tan (C) measured from PDRN formulations over the frequency range of 0.1–80 rad/s.
Figure 6. Osmolality measured from PDRN formulations. Asterisk represents tolerable tonicity range for the eye illustrated in this figure was referred to from a research literature published22.
Only in the case sodium chloride used as an isotonic agent, properties of sample were turned into packed gel and great changes in G0 and G00 were observed. Therefore it is reasonable to use D-glucose or D-sorbitol, instead of sodium chloride, as isotonic agents for ophthalmic PDRN formulations. Among the thickeners, PVP was found to be an ideal thickener because carbopol 940 was found to interact with PDRN and induced molecular structure changes while HPMC 2910 was unsuitable because foaming was observed when dissolved in water. Among PVP K30 and PVP K90, PVP K90 showed more increase in viscoelastic moduli of PDRN formulations while both had similar influence on osmotic pressure of the formulations when used at similar amounts. Thus, PVP K90 can be more favorable to be used as a thickener, considering its effects on the viscoelastic properties of PDRN solution. Benzalkonium chloride (at concentrations 50.1% w/v) can also be included into the ophthalmic formulations as an anti-bacterial preservative.
DOI: 10.3109/03639045.2015.1040417
Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article. This research was supported by the Chung-Ang University Research Scholarship Grants in 2014.
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