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Local treatment of the inner ear: A study of three different polymers aimed for middle ear administration ab
ac
ab
d
Cecilia Engmér Berglin , Pernilla Videhult Pierre , Andreas Ekborn , Tobias Bramer , d
Katarina Edsman , Malou Hultcrantz
ab
& Göran Laurell
c
a 1
Department of Clinical Science, Intervention, and Technology, Karolinska Institutet, Stockholm, Sweden b 2
Department of Otorhinolaryngology, Karolinska University Hospital, Stockholm, Sweden
c 3
Department of Surgical Sciences
d 4
Department of Pharmacy, Uppsala University, Uppsala, Sweden Published online: 06 Jul 2015.
To cite this article: Cecilia Engmér Berglin, Pernilla Videhult Pierre, Andreas Ekborn, Tobias Bramer, Katarina Edsman, Malou Hultcrantz & Göran Laurell (2015): Local treatment of the inner ear: A study of three different polymers aimed for middle ear administration, Acta Oto-Laryngologica To link to this article: http://dx.doi.org/10.3109/00016489.2015.1058534
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Acta Oto-Laryngologica. 2015; Early Online, 1–10
ORIGINAL ARTICLE
Local treatment of the inner ear: A study of three different polymers aimed for middle ear administration
CECILIA ENGMÉR BERGLIN1,2, PERNILLA VIDEHULT PIERRE1,3, ANDREAS EKBORN1,2, TOBIAS BRAMER4, KATARINA EDSMAN4, MALOU HULTCRANTZ1,2 & GÖRAN LAURELL3 Downloaded by [University of Otago] at 05:50 13 July 2015
1
Department of Clinical Science, Intervention, and Technology, Karolinska Institutet, Stockholm, Sweden, Department of Otorhinolaryngology, Karolinska University Hospital, Stockholm, Sweden, 3Department of Surgical Sciences and 4Department of Pharmacy, Uppsala University, Uppsala, Sweden
2
Abstract Conclusion: A formulation based on sodium hyaluronate (NaHYA) was the most promising candidate vehicle for intra-tympanic drug administration regarding conductive hearing loss, inflammatory reactions, and elimination. Objectives: Recent advances in inner ear research support the idea of using the middle ear cavity for drug administration to target the inner ear. This paper presents rheological and safety assessments of three candidate polymer formulations for intra-tympanic drug administration. Method: The formulations were based on sodium carboxymethyl cellulose (NaCMC), sodium hyaluronate (NaHYA), and poloxamer 407 (POL). Rheological studies were performed with a controlled rate instrument of the couette type. Safety studies were performed in guinea pigs subjected to an intra-tympanic injection of the formulations. Hearing function was explored with ABR before and 1, 2, and 3 weeks after the injection. Elimination of the formulations marked with coal was explored with an endoscopic digital camera 1, 2, and 3 weeks after injection. Middle and inner ear morphology was examined with light microscopy 6 days after injection. Results: The results speak in favor of NaHYA, since it did not cause prolonged hearing threshold elevations. The results of the elimination and morphological investigations support the conclusion of NaHYA being the most promising candidate for intra-tympanic administration.
Keywords: ABR, carboxymethyl cellulose, guinea pig, hyaluronan, intra-tympanic, morphology, poloxamer 407, polymer, targeted drug delivery, toxicity
Introduction Topical application of drugs is used to treat a variety of sites and organs, such as the eye and the skin. Recent advances in inner ear research support the idea of using the middle ear cavity as a reservoir for local drug administration to target cells in inner ear compartments [1]. Destructive treatment of vertigo with gentamicin in Ménière’s disease is today the only established clinical method that employs intra-tympanic drug administration [1]. In the literature, numerous studies can also be found on intra-tympanic injection of glucocorticoids to treat
different inner ear disorders in humans, such as autoimmune inner ear disease, idiopathic sudden sensorineural hearing loss, tinnitus, or Ménière’s disease, but data supporting their efficacy are still weak [1]. There are a number of advantages of middle ear drug delivery over systemic administration. With local drug delivery, unacceptable side-effects and drug interactions in the blood compartment may be avoided. Moreover, the drug uptake to inner ear compartments may be increased, since these compartments can be difficult to reach from the systemic circulation due to different barrier systems.
Correspondence: Professor Göran Laurell, MD, Uppsala University Hospital, ENT Clinic, SE-751 85 Uppsala, Sweden. Tel: +46 18 6115363. Fax: +46 18 558231. E-mail: [email protected]
(Received 9 April 2015; accepted 25 May 2015) ISSN 0001-6489 print/ISSN 1651-2251 online 2015 Informa Healthcare DOI: 10.3109/00016489.2015.1058534
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The round window membrane is considered to be the most important route for a drug to reach the inner ear when administered intra-tympanically [1], although recent data suggest that the oval window can also be of importance [2]. Therefore, a key factor for drug uptake is the contact time between the drug and this/these membrane/membranes [1]. At present, patients subjected to intra-tympanic drug administration are, therefore, often requested to remain still for about half an hour with the injected ear facing upwards. This is inconvenient, especially if both ears need to be treated at the same time. The problem may be circumvented by using a polymer; the inclusion of a polymer in a drug formulation can improve the mucoadhesive [3] and rheological [4] properties of the formulation, resulting in a prolonged residence time at the site of the absorption and, thereby, a facilitated drug uptake. A semisolid polymer formulation can be in the form of e.g. a gel or a polymer solution. A polymer gel usually consists of a polymer network formed by chemical cross-links or physical interactions [5]. Most gels are soft, solid or solid-like materials consisting of at least two components, one of which is a liquid present in abundance [5]. If a polymer network is lacking, the formulation becomes fluid and is usually classified as a polymer solution [5]. The solidity of a polymer formulation is dependent on its viscoelastic properties. The viscosity is a critical factor of an intra-tympanically applied formulation aimed for treatment of inner ear disorders, since it can be expected that a formulation with a low viscosity will not closely stay in contact with the round and/or oval window membranes and, therefore, most probably give a low drug transport to the inner ear. On the other hand, if the viscosity is too high, the formulation might be hard to eliminate from the middle ear and lead to a conductive hearing loss for a prolonged period of time. Little is known about the elimination mechanisms of an intra-tympanically applied polymer formulation, but Eustachian tube mucociliary transport is likely a fundamental mechanism [6]. The inner ear structures are delicate and research has to be undertaken to develop not only effective but also safe local drug delivery systems in search for potential and novel models. The objective of the experimental studies presented in this paper was to explore three different polymer formulations as candidate vehicles for intra-tympanic drug administration. The explored polymers were sodium carboxymethyl cellulose, sodium hyaluronate, and poloxamer 407. These polymers are already used in different pharmaceutical formulations and are considered non-toxic and non-irritant [7]. Sodium carboxymethyl cellulose is a widely employed
viscosity modifier and emulsifier [7]. It is used, e.g., in artificial tears and as a food additive [7]. Sodium hyaluronate is a linear molecule composed of a repeating disaccharide of N-acetyl-D-glucosamine and D-glucuronic acid [8]. It is naturally and ubiquitously occurring in humans and a major component of the extracellular matrix [8]. It is produced by hyaluronan synthase, is degraded by hyaluronaidases, and is immunologically inert [9]. Poloxamer 407 is a polyoxyethylene polyoxypropylene block copolymer used in several different pharmaceutical preparations, for both internal and external use [7]. First, the rheological properties of the polymer formulations were investigated in vitro. Second, the polymer formulations were administered into the middle ear of guinea pigs and their effects on the electrophysiological hearing thresholds and morphology as well as their elimination were investigated. Materials and methods Animals A total of 35 guinea pigs (Duncan-Hartley) of both sexes were purchased from a local breeder and were maintained on a 12:12 h light/dark cycle with unrestricted access to food and water. The care and use of the animals were approved by the local animal care and use committee, Stockholms Norra Djurförsöksetiska Nämnd (N 334/05), according to the Declaration of Helsinki. Chemicals Sodium carboxymethyl cellulose (molecular weight: 100 kDa; Cekol100 000), linear sodium hyaluronate (approximate molecular weight: 4000 kDa; Healon), and poloxamer 407 (average molecular weight: 12 kDa; Lutrol F 127) were generous gifts from Noviant B.V. (Nijmegen, The Netherlands), Pharmacia Upjohn (Uppsala, Sweden), and BASF Aktiengesellschaft (Ludwigshafen, Germany), respectively. Ketamine (Ketalar, 50 mg/mL, Pfizer AB, Täby, Sweden) and xylazin (Rompun vet, 20 mg/mL, Bayer Health Care AG, Leverkusen, Germany) were used for general anesthesia and lidocaine (Xylocain, 20 mg/mL, AstraZeneca, Södertälje, Sweden) for local anesthesia. Preparation of experimental polymer formulations Experimental polymer formulations were prepared by mixing commercial polymer products with different solvents. A summary is given in Table I. A sodium carboxymethyl cellulose formulation (NaCMC) was
Three polymers
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Table I. Experimental polymer formulations were prepared by mixing commercial polymer products with different solvents. Final concentration (% w/w)
Abbreviation of experimental polymer formulation
0.5
NaCMC
~7.2
0.5
NaHYA
~7.2
25
POL
Chemical name
Brand name
Solvent
Final pH
Sodium carboxy-methyl cellulose
Cekol
Saline
7.3–7.5
Sodium hyaluronate
Healon
Phosphate-buffered saline
Poloxamer 407
Lutrol
Phosphate-buffered saline
prepared by dispersing Cekol in saline (9 mg/ml) and allowing it to completely dissolve before pH adjustment with sodium hydroxide. Additional saline was then added to a final polymer concentration of 0.5% (w/w). A sodium hyaluronate formulation (NaHYA) was prepared by mixing Healon (1% w/v) with phosphate-buffered saline to a final polymer concentration of 0.5% (w/w). A poloxamer 407 formulation (POL) was prepared by dissolving Lutrol in phosphate-buffered saline to a final poloxamer 407 concentration of 25% (w/w). For semiquantification of the elimination of the formulations, NaCMC, NaHYA, and POL, respectively, were mixed (5:95, v/v) with a coal suspension (a kind gift from the Karolinska Pharmacy, Stockholm, Sweden). All polymer formulations were autoclaved before being administered to the animals.
subjected to an injection of NaCMC, NaHYA, or POL (volume = 0.15 mL; needle: BD Microlance 30G, external diameter = 0.3 mm) into the auditory bulla through the skin of the auricle after paracentesis of the tympanic membrane. An operating microscope was used to determine the point of injection and to observe the flow of the polymer formulation into the middle ear cavity. The animals were allowed to regain consciousness prior to being returned to the vivarium. The effects and safety of the intra-tympanically administered polymer formulations were studied by hearing assessment using acoustically evoked ABR, by visual semi-quantification of elimination of the polymer formulations marked with coal after opening the auditory bulla, and by light microscopic examination of the middle and inner ears. Acoustically evoked ABR assessment
In vitro experiments NaCMC, NaHYA, and POL were investigated rheologically with a Bohlin VOR Rheometer (Bohlin Reologi, Lund, Sweden), a controlled rate instrument of the couette type. Viscosimetric measurements were carried out on NaCMC and NaHYA at 37 C, using a concentric cylinder system (C8). Oscillating measurements were performed on POL with the same system at 20, 25, 30, 35, and 40 C. The rheological properties of the coal-marked formulations were not examined. In vivo experiments The animals were ~10 weeks old at the start of the experiment. They were anesthetized with ketamine (40 mg/kg b.w., i.m.) and xylazine (12 mg/kg b.w., i.m.). Additional doses of ketamine and xylazine were administered when needed to maintain an adequate depth of anesthesia. The ear canals, tympanic membranes, and electrophysiological hearing thresholds of the animals were verified as being normal by otoscopic examination and by assessment of acoustically evoked auditory brainstem response (ABR; for details, see below). While still under anesthesia, the animals were
Fifteen albino guinea pigs (mean weight = 303 g; weight range = 269–345 g) were used to evaluate the effects of the polymer formulations on acoustically evoked ABR thresholds. The measurements were performed with a TDT system II (Tucker Davies Technologies, Gainesville, FL), a standard modular system operated via a personal computer, using TDT BIO SIG ver. 2.0 software. The sound stimuli were delivered inside the ear canal through a speculum from a TDT high frequency transducer with the animal placed in a soundproof box. The auditory stimuli consisted of 2 ms full sine wave tone bleeps at 3, 6, 12, 20, and 30 kHz designed with a cosine gate and were delivered to the ear at a rate of 30/s. The sound stimulus intensity was varied in 5 dB intervals. The electric signals were collected by stainless steel electrodes placed subcutaneously in the infraauricular region of the measured ear and in the vertex. The reference electrode was placed on the nasal bridge. The signal was amplified and sampled over a 10 ms period, digitally averaged over 2000 cycles, low-pass altered, and presented as an electroencephalogram on the computer screen. The electrophysiological hearing threshold was defined as the lowest intensity at which a reproducible response could be recorded.
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The 15 animals were randomized to be administered NaCMC, NaHYA, or POL into the middle ear cavity bilaterally (n = 5 in each group). ABR assessments were repeated weekly for 3 weeks after the administration. Thresholds were considered elevated if being at least 10 dB higher compared to the baseline value at more than one frequency. After the final ABR assessment, the animals were decapitated while still under deep anesthesia.
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Visual semi-quantification of coal-marked polymer formulation by opening the bulla Nine albino guinea pigs (mean weight = 279 g; weight range = 220–320 g) were used to evaluate the elimination of NaCMC, NaHYA, or POL marked with coal. The animals were randomized into three groups (n = 3 in each group). Each ear of the animals in the different groups was randomized to be administered NaCMC, NaHYA, or POL marked with coal in such a way that no animal received the same polymer formulation in both ears. One animal in each group was re-anesthetized 1, 2, or 3 weeks after the administration. The auditory bullae were opened with a dorsolateral approach. All ears were examined for remnants of the coal-marked polymer formulations and inflammatory response in the middle ear, using a dissecting microscope. Quantification was performed with a four-graded scale (Table II). The middle ears were photographed using an endoscopic digital camera. Finally, the animals were euthanized as described above. Table II. Guinea pigs were subjected to an intra-tympanic injection (0.15 mL) of a polymer formulation of sodium carboxymethyl cellulose (0.5% w/w; NaCMC), sodium hyaluronate (0.5% w/w; NaHYA), or poloxamer 407 (25% w/w; POL) marked with coal. The auditory bullae were opened 1, 2, or 3 weeks after the injection. The signs of remnants of the coal-marked formulations and inflammatory response in the middle ear was quantified using a four-graded scale. Stage
Criteria
A
No visible signs of NaCMC, NaHYA, POL, or coal; no visible signs of inflammation.
B
Visible coal remnants in the middle ear mucosa; no visible signs of NaCMC, NaHYA, POL, or liquid in the middle ear cavity, around the ossicules, or at the round window; no edema in the middle ear mucosa.
C
Visible coal remnants around the ossicules and/or at the round window; edema in or reddened middle ear mucosa or liquid in the middle ear cavity.
D
Middle ear filled with NaCMC, NaHYA, or POL marked with coal.
Light microscopic examination of the middle and inner ears Eleven albino guinea pigs (mean weight = 284 g; weight range = 242–327 g) were used for light microscopic evaluation of inflammatory reactions in the tympanic membrane, the middle ear mucosa, and the round window membrane after intra-tympanic administration of NaCMC, NaHYA, or POL. Light microscopic examination of the hair cells and stria vascularis of the basal turn of the cochlea, close to the round window membrane, was also performed. The animals were randomized to be administered NaCMC, NaHYA, or POL into the middle ear bilaterally (n = 3 in each group). One animal was injected with saline (9 mg/ml) and one received no injection, serving as a control. Six days after the administration, the animals were re-anesthetized and euthanized by decapitation. The auditory bullae were disassembled from the temporal bone and fixed in paraformaldehyde (4%) for 2 h. They were stored in paraformaldehyde (0.5%) at 4 C until decalcified with EDTA (0.1 M in phosphate buffer (0.1 M)), washed in phosphate buffer (0.1 M) several times, dehydrated in a graded ethanol series, and embedded in JB4 resin (Polyscience). Then, they were sectioned at 3-mm thickness with a rotary microtome (Microm HM 355 S). The left ears were sectioned perpendicularly to the RMW and across the cochlea. The right ears were sectioned perpendicularly both to the round window membrane and the tympanic membrane across the middle ear and the cochlea. Every 10th section was mounted on glass slides, stained with toluidine blue (0.1%), and examined under a light microscope (Zeiss) equipped with a digital camera (Altra 20 soft imaging system, Olympus). The thickness of the round window membrane and the tympanic membrane was measured using image analysis software (Cell, Olympus). Measurement of three adjacent sections of each round window membrane and tympanic membrane was made at the mid-portion of the round window membrane and tympanic membrane, respectively, and the value of the thickest part was chosen as a representative value of inflammatory reaction. Data analysis The Wilcoxon signed-rank test was used to compare the median electrophysiological hearing thresholds obtained before the injection of a specific polymer formulation with those obtained after the injection. Kruskal-Wallis and Dunn’s multiple comparison tests were used to compare the hearing thresholds of independent groups. A p-value < 0.05 was
Three polymers considered statistically significant. All statistical tests were two-sided. When the results of an ABR measurement showed that the mean electrophysiological hearing threshold difference of the different frequencies between the two ears of one animal was more than 20 dB, the ear with the lowest threshold was excluded to reduce the risk of erroneous hearing thresholds due to bone conduction of the sound stimuli to the contralateral ear. Results
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In vitro experiments The rheological investigation showed that NaCMC and NaHYA were high viscosity solutions rather than gels (Figure 1). At the shear rates studied, NaCMC exhibited a shear thinning behavior, whereas NaHYA did not (Figure 1). POL formed a semi-solid gel and its rheology changed drastically at a temperature of ~15 C (Figure 2). POL fulfilled a rheological definition of a gel, which is having a frequency independent elastic (G’) modulus and a much higher elastic than viscous (G’’) modulus over a large frequency range (Figure 3) [5,10]. In vivo experiments
5
NaCMC injection, in four animals 2 weeks after NaCMC injection, and in three animals 3 weeks after NaCMC injection. In the NaHYA group, the corresponding figures were two animals 1 week after NaHYA injection, one animal 2 weeks after NaHYA injection, and one animal 3 weeks after NaHYA injection. In the POL group, the ear with the lowest mean threshold was excluded in one animal 3 weeks after POL injection. Ears injected with NaHYA had significantly lower threshold shifts at all evaluated frequencies 1 week after the administration compared to ears injected with POL or NaCMC, at 20 and 30 kHz 2 weeks after the administration compared to ears injected with POL, at 30 kHz 2 weeks after the administration compared to ears injected with NaCMC, and at 12 and 30 kHz 3 weeks after the administration compared to ears injected with NaCMC. There were no significant differences between NaCMCand POL-injected ears.
Visual semi-quantification of coal-marked polymer formulation by opening the bulla The results of the visual semi-quantification of elimination of NaCMC, NaHYA, and POL marked with coal are shown in Table III. As can be seen, in no single ear was there a total elimination of coal. Figure 5
Acoustically evoked ABR assessment. The results of the acoustically evoked ABR assessments are illustrated in Figure 4. Due to an intra-individual side difference in mean electrophysiological hearing threshold exceeding 20 dB, the ear with the lowest mean threshold was excluded in one animal 1 week after
Figure 1. Formulations of sodium carboxymethyl cellulose (0.5% w/w; NaCMC) and sodium hyaluronate (0.5% w/w; NaHYA) were prepared and investigated rheologically. The results of the viscosimetric measurements for NaCMC (closed triangles) and NaHYA (closed squares) at 37 C using a concentric cylinder system (C8) are shown.
Figure 2. A formulation of poloxamer 407 (25% w/w; POL) was prepared and investigated rheologically at 10, 15, 20, 25, 30, 35, and 40 C, using oscillating measurements. The viscoelastic properties of POL at 1 Hz are shown. G¢ and G¢¢ signify the elastic and viscous moduli, respectively.
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Figure 3. The elastic (G’; closed circles) and viscous (G’’; open circles) moduli of POL from an oscillating measurement at 30 C.
shows photographs from a middle ear of stage B (5A and 5B) and stage C (5C and 5D). Light microscopic examination of the middle and inner ears The results of the light microscopic examination of morphological signs of inflammation in the tympanic membrane and the round window membrane are shown in Table IV. Due to technical problems, the number of observations differed between groups. The thickness of the round window membrane in normal, unexposed control ears was close to 10 mm, which is in agreement with previous results [11]. Remnants of the polymer formulation and a swollen mucosa could be seen in the middle ear of animals injected with NaCMC (Figures 6A and B). The mucosa was also swollen in POL-injected ears, but no remnants of the
polymer formulation could be seen. In ears injected with NaHYA, there were neither any signs of inflammation in the mucosa nor any remnants of the polymer formulation (Figure 6C). The hair cells and the stria vascularis of the cochlea, close to the round window membrane, could be evaluated in three ears in the NaHYA group and in two ears in the POL and NaCMC groups, respectively. There were no signs of damage in any specimen. Discussion This paper presents rheological and safety assessments of three different polymer formulations aimed for intra-tympanic drug delivery. The results speak in favor of NaHYA. Especially, in contrast to NaCMC and POL, NaHYA did not cause prolonged hearing
Figure 4. Guinea pigs (n = 15) were subjected to an intra-tympanic injection (0.15 mL) of NaCMC (A), NaHYA (B), or POL (C) bilaterally (n = 5 in each group). Acoustically evoked auditory brainstem response (ABR) assessment was performed before as well as 1, 2, and 3 weeks after the injection. The graphs show the electrophysiological hearing threshold shifts, i.e. the difference between the hearing thresholds obtained before and after (1 week, open circles; 2 weeks, closed circles; 3 weeks; open triangles) the injection. Data are expressed as mean ± SEM. The measured frequencies, 3, 6, 12, 20, and 30 kHz are center frequencies of the presented auditory stimuli.
7
Three polymers
Table III. Guinea pigs (n = 9) were subjected to an intra-tympanic injection (0.15 mL) of NaCMC (n = 6 ears), NaHYA (n = 6 ears), or POL (n = 6 ears) marked with coal. Their auditory bullae were opened 1, 2, or 3 weeks after the injection and the middle ears were photographed with an endoscope and digital camera. The photographs were used for semi-quantification of the elimination of the coal-marked polymer formulations. Details of the different stages are given in Table II. Absolute numbers of ears are given. Week 1
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Stage
Week 2
Week 3
NaCMC
NaHYA
POL
NaCMC
NaHYA
POL
NaCMC
NaHYA
POL
A
0
0
0
0
0
0
0
0
0
B
0
1
1
0
0
0
0
2
1
C
2
1
1
2
2
2
2
0
1
D
0
0
0
0
0
0
0
0
0
threshold elevations. The results of the elimination and morphological investigations support the conclusion of NaHYA being the most promising candidate for intra-tympanic administration.
Figure 5. Guinea pigs (n = 9) were subjected to an intra-tympanic injection (0.15 mL) of NaCMC (n = 6 ears), NaHYA (n = 6 ears), or POL (n = 6 ears) marked with coal. Photographs of a middle ear injected with coal-marked NaHYA (A and B) and NaCMC (C and D) 3 weeks earlier are shown. (A) A minor area of coal remnant can be seen through the tympanic membrane to the right of the handle of the malleus. (B) The middle ear cavity from a dorso lateral view after the bulla has been opened. Coal remnants can be seen in the mucosa on the inside of the tympanic membrane (TM) and at the round window niche (arrow). There are no visible signs of inflammation or remnants of the formulation in the middle ear. The open black circle indicates the apex of the cochea. (C). The tympanic membrane is thickened and with liquid in the middle ear. (D) The middle ear cavity from a dorso lateral view after the bulla has been opened. Coal remnants can be seen in the mucosa, especially on the tympanic membrane (TM) and at the round window niche (arrow). Coal-marked formulation surrounds the ossicules between the tympanic membrane and the cochlea. The open black circle indicates the apex of the cochlea.
Electrophysiological examination of the hearing using acoustically evoked ABR is a very common method in experimental hearing research. When the sound stimuli is delivered through the ear canal, as in the present study, the hearing thresholds are expected to be elevated when something resides in the middle ear, obstructing the transmission of sound, i.e. causing a conductive hearing loss. Elevated thresholds can also be due to induced inflammatory reactions causing e.g. changes to the tympanic membrane and toxic effects to the inner ear. The fact that there were no significant threshold shifts in the ears injected with NaHYA implies that most of NaHYA had been eliminated from the middle ear already 1 week after the injection, that its induced inflammatory reactions were reversible, and that NaHYA was not ototoxic. Coal in the middle ear was easily visualized using an endoscopic digital camera. However, our interpretation of the results with the coal-marked polymer formulations is only tentative. It has to be pointed out that coal is not soluble in NaCMC, NaHYA, or POL. Therefore, the fact that in no single ear was there a total elimination of coal remnants does not prove that remnants of NaCMC, NaHYA, or POL were still present in the middle ear. In a previous experimental study on a non-soluble form of a drug in a poloxamer 407-based gel, it was observed that drug microparticulates were present in the middle ear beyond the disappearance of the gel matrix [12]. Furthermore, in the present investigation, the number of observations was few in the coal experiments, which makes it impossible to draw any firm conclusions from this part of the study. However, the results point in the same direction as the results of the ABR assessments. For example, when coal remnants were found around the middle ear ossicles or liquid in the middle ear cavity was apparent (the criteria of Stage C), as in all NaCMC-administered ears 1, 2, and 3 weeks after the administration, effects on the hearing function can be expected, which were also the case in the majority of the NaCMC-administered ears.
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Table IV. The thickness (mm) of the round window membrane and tympanic membrane obtained from guinea pigs 6 days after a bilateral intra-tympanic injection (0.15 mL) of NaCMC (n = 3 ears), NaHYA (n = 3 ears), POL (n = 3 ears) or saline (9 mg/ml; n = 1 ear). Each value represents one sample. NaCMC
NaHYA
POL
Saline
No treatment
Round window Tympanic Round window Tympanic Round window Tympanic Round window Tympanic Round window Tympanic 26
45
14.2
18.2
20.8
54.9
19.6
24.1
13.3
17.9
23.1
54.2
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18.2
The results of the coal experiments support the ABR findings as well as the light microscopy investigation, indicating that NaHYA is the best of the three studied polymer formulations for intratympanic administration. Sodium hyaluronate, which was used for preparation of NaHYA, is the sodium salt of hyaluronic acid. In the literature, hyaluronan is a general term for this polymer, irrespective of its degree of dissociation. Locally applied exogenous hyaluronan has been used for a long time in otological research, both in experimental animals [11,13,14] and humans [15,16]. These studies have shown that it is welltolerated and not ototoxic, even when administered into the middle ear when there has been a direct access to the inner ear [13,14]. The light microscopic investigation described in this paper showed that NaHYA induced less thickening of the round window membrane and tympanic membrane than NaCMC and POL. The effect of NaHYA on the round window membrane agrees with that of a previous study, in which a similar hyaluronan formulation was used [11]. In that study, the thickness of the round window
22.6
30.5
10.1
13.2
10
45.4
membrane had returned to normal values after 4 weeks. Exogenous hyaluronan is eliminated primarily via the Eustachian tube [6], which is a general mechanism fundamental for elimination of products from the middle ear cavity. It may be totally eliminated within a day in the rat, according to an experimental study using a formulation with a hyaluronan concentration twice that of the present investigation [6]. The results of our study using NaHYA marked with coal suggest that a hyaluronan-based formulation may stay in the middle ear considerably longer in the guinea pig. However, there were no statistically significant hearing threshold shifts 1, 2, and 3 weeks after administration of NaHYA. Neither were there any remnants of NaHYA found in the light microscopic investigation 6 days after its administration. Obviously, further studies are needed if the elimination kinetics of NaHYA from the middle ear is to be established. Diverging data on the elimination of exogenous hyaluronan from the middle ear cavity may be due to differences in transport mechanisms in different species, administered volume, and concentration as well as molecular weight of hyaluronan.
Figure 6. Guinea pigs (n = 11) were subjected to a bilateral intra-tympanic injection of NaCMC (n = 3), NaHYA (n = 3), POL (n = 3), saline (n = 1), or no treatment (n = 1). After 6 days, the animals were euthanized for histological examination of the middle and inner ears using light microscopy. A NaCMC-treated (A and B) and a NaHYA-treated (C) middle ear is shown. (A) A submucosal edema can be observed in proximity to remnants of the formulation (arrow) in the middle ear cavity (ME). (B) The round window membrane (RWM) is thickened and the polymer formulation can be seen between the round window membrane and the stapes (bone in top of the picture) in the middle ear cavity (ME). (C) A NaHYA-treated ear sectioned perpendicularly both to the round window membrane (RWM) and the tympanic membrane (TM) across the middle ear cavity (ME) and the cochlea. The round window membrane and the tympanic membrane are slightly thickened, but the mucosa of the middle ear shows no sign of inflammatory reaction. The organ of Corti (OoC) and stria vascularis (SV) in the basal part of the cochlea appear normal.
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Three polymers In the present study, the hyaluronan concentration was not varied, but it is well known that its viscosity is directly proportional to its concentration [8]. As a result, the elimination rate of a hyaluronan formulation from the middle ear cavity will most likely be reduced when the concentration of hyaluronan is increased. Similarly, increasing the molecular weight of hyaluronan will increase the viscosity of the formulation [8]. The molecular weight of hyaluronan depends on the hyaluronan source and on the isolation and purification procedures used [8]. In the present study, all formulations were autoclaved prior to being used in vivo. Hyaluronan has been shown to be sensitive to high temperatures [8]. Bothner Wik [17] has suggested how the average molecular weight (Mw) of hyaluronan can be calculated from a known zero shear viscosity (h0) and concentration (c) [17]. Equation (1) is valid when h0 < 0.01 Pa s, whereas equation (2) is valid when h0 > 0.1 Pa s.
h0 = 1.7 × 10−7 × (c × Mw )
0.74
h0 = 5.3 × 10−26 × (c × Mw )
3.6
(1)
(2 )
Unfortunately, h0 of the hyaluronan formulation used in this study was determined to 0.085 (Figure 1), which means that it lies in a region where the average molecular weight is difficult to calculate using either of these two equations. However, using equation (2) allows us to at least estimate the average molecular weight to ~1000 kDa, indicating a massive degradation of NaHYA following heat sterilization. As illustrated in Figure 1, NaCMC was pseudoplastic, which means that the carboxymethyl cellulose molecules deformed and aligned in the streamlines of flow, resulting in a decrease in the viscosity. This behavior occurs when pushing a pseudoplastic formulation through medical needles, which makes the needles easy to handle. In contrast, NaHYA did not exhibit a shear thinning behavior (Figure 1). Nonetheless, hyaluronan has been shown to be pseudoplastic when applying a higher shear force or when it is less concentrated [8]. Even though sodium carboxymethyl cellulose is considered non-toxic and non-irritant, there are few published studies on the use of sodium carboxymethyl cellulose in otological research, in contrast to hyaluronan. The results of the present study show that NaCMC might be less convenient for intra-tympanic administration than NaHYA. In fact, sodium carboxymethyl cellulose might even be ototoxic, according to a guinea pig study [18]. However, in that study, a commercial
9
formulation aimed for nasal application was used and it is unknown whether the hearing loss was caused by sodium carboxymethyl cellulose itself or by some other component or property of the formulation. Poloxamers are temperature-responsive, being less viscous when refrigerated or at room temperature than at body temperature. To facilitate the intratympanic injection in the present study, samples of POL were taken from the refrigerator immediately prior to the injection. Yet, the viscosity of POL was still high and, to avoid excess trauma to the tympanic membrane, POL as well as NaCMC and NaHYA were injected through the auditory bulla. Similar to sodium carboxymethyl cellulose and hyaluronan, poloxamer 407 is considered non-toxic and nonirritant [7]. Nonetheless, the results of the present study imply that POL is less suitable for intratympanic administration than NaHYA. In particular, POL induced a striking thickening of the tympanic membrane and substantial effects on the hearing thresholds. However, according to previous publications, poloxamer 407 may be well-tolerated also when administered into the middle ear [12,19]. In a study, most of the conductive hearing loss had disappeared ~4 weeks after intra-tympanic administration of poloxamer 407 [12], and several weeks earlier in another study [19]. In these studies, a lower concentration of poloxamer 407 and a smaller injected volume were used, which might explain their more promising results. A variety of delivery systems has been investigated in order to achieve a reproducible and prolonged drug exposure of the inner ear. These include repeated transtympanic injections, instillation through ventilation tubes, and continuous infusion through microcathethers. The method of a single transtympanic injection of a polymer formulation involves no surgery and only minor trauma. Its major drawback is that it induces a transient conductive hearing loss and, thereby, it may not be tolerated by humans. Several experimental animal studies have explored hyaluronan formulations as a vehicle for intra-tympanic administration of pharmacological compounds [11,20]. There are even some clinical studies, in which dexamethasone was administered into the middle ear in a hyaluronan-based formulation [15,16]. The study presented in this paper supports further investigation of hyaluronan for middle ear administration. It also suggests cautiousness regarding middle ear administration of sodium carboxymethyl cellulose, especially since it has been proposed to be ototoxic [18]. Finally, the results using poloxamer 407 were not promising, but may have been due to a too high concentration, since other researchers have
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shown good results when using less concentrated poloxamer 407-based formulations.
Acknowledgments
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The authors acknowledge Staffan Eksborg for the statistical analyses and AFA, the Acta OtoLaryngologica Foundation, and Rosa and Emanuel Nachmansson’s Foundation for their generous financial support for the conduct of the research and preparation of the article. The foundations had no involvement in the study design, in the collection, analysis, and interpretation of data, in the writing of the report nor in the decision to submit the paper for publication. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
[8]
[9]
[10] [11]
[12]
[13]
[14]
[15]
References [16] [1] Salt AN, Plontke SK. Principles of local drug delivery to the inner ear. Audiol Neurootol 2009;14:350–60. [2] King EB, Salt AN, Kel GE, Eastwood HT, O’Leary SJ. Gentamicin administration on the stapes footplate causes greater hearing loss and vestibulotoxicity than round window administration in guinea pigs. Hear Res 2013;304: 159–66. [3] Edsman K, Hagerstrom H. Pharmaceutical applications of mucoadhesion for the non-oral routes. J Pharm Pharmacol 2005;57:3–22. [4] Carlfors J, Edsman K, Petersson R, Jornving K. Rheological evaluation of Gelrite in situ gels for ophthalmic use. Eur J Pharm Sci 1998;6:113–19. [5] Almdal K, Dyre J, Hvidt S, Kramer O. Towards a phenomenological definition of the term ’gel’. Polym Gels Netw 1993;1:5–17. [6] Laurent C, Hellstrom S, Appelgren LE, Sehlin J, Lilja K. Fate of exogenous hyaluronic acid in the middle ear. Am J Otolaryngol 1986;7:334–40. [7] Wade A, Weller PJ. Handbook of pharmaceutical excipients. In Wade A, Weller PJ, editors. Washington & London:
[17]
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American Pharmaceutical Association and The Pharmaceutical Press; 1994. Lapcik L Jr, Lapcik L, De Smedt S, Demeester J, Chabrecek P. Hyaluronan: Preparation, Structure, Properties, and Applications. Chem Rev 1998;98:2663–84. Bastow ER, Byers S, Golub SB, Clarkin CE, Pitsillides AA, Fosang AJ. Hyaluronan synthesis and degradation in cartilage and bone. Cell Mol Life Sci 2008;65:395–413. Ross-Murphy SB, McEvoy H. Fundamentals of hydrogels and gelation. Br Polym J 1986;18:2–7. Saber A, Laurell G, Bramer T, Edsman K, Engmér C, Ulfendahl M. Middle ear application of a sodium hyaluronate gel loaded with neomycin in a Guinea pig model. Ear Hear 2009;30:81–9. Piu F, Wang X, Fernandez R, Dellamary L, Harrop A, Ye Q, et al. OTO-104: a sustained-release dexamethasone hydrogel for the treatment of otic disorders. Otol Neurotol 2011;32:171–9. Bagger-Sjoback D. Sodium hyaluronate application to the open inner ear: an ultrastructural investigation. Am J Otol 1991;12:35–9. Laurent C, Anniko M, Hellstrom S. Hyaluronan applied to lesioned round window membrane is free from cochlear ototoxicity. Acta Otolaryngol 1991;111:506–14. Gouveris H, Selivanova O, Mann W. Intratympanic dexamethasone with hyaluronic acid in the treatment of idiopathic sudden sensorineural hearing loss after failure of intravenous steroid and vasoactive therapy. Eur Arch Otorhinolaryngol 2005;262:131–4. Selivanova OA, Gouveris H, Victor A, Amedee RG, Mann W. Intratympanic dexamethasone and hyaluronic acid in patients with low-frequency and Meniere’s-associated sudden sensorineural hearing loss. Otol Neurotol 2005;26: 890–5. Bothner Wik H. Rheological studies of sodium hyaluronate in pharmaceutical preparations [Doctoral thesis]. Uppsala: Uppsala University; 1991. Antonelli PJ, Sampson EM, Lang DM. Safety and efficacy of carbomethylcellulose foam in guinea pig middle ear surgery. Otolaryngol Head Neck Surg 2010;142:405–8. Wang X, Dellamary L, Fernandez R, Harrop A, Keithley EM, Harris JP, et al. Dose-dependent sustained release of dexamethasone in inner ear cochlear fluids using a novel local delivery approach. Audiol Neurootol 2009;14: 393–401. Berglin CE, Pierre PV, Bramer T, Edsman K, Ehrsson H, Eksborg S, et al. Prevention of cisplatin-induced hearing loss by administration of a thiosulfate-containing gel to the middle ear in a guinea pig model. Cancer Chemother Pharmacol 2011;68:1547–56.
Acta Oto-Laryngologica Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/ioto20
Local treatment of the inner ear: A study of three different polymers aimed for middle ear administration ab
ac
ab
d
Cecilia Engmér Berglin , Pernilla Videhult Pierre , Andreas Ekborn , Tobias Bramer , d
Katarina Edsman , Malou Hultcrantz
ab
& Göran Laurell
c
a 1
Department of Clinical Science, Intervention, and Technology, Karolinska Institutet, Stockholm, Sweden b 2
Department of Otorhinolaryngology, Karolinska University Hospital, Stockholm, Sweden
c 3
Department of Surgical Sciences
d 4
Department of Pharmacy, Uppsala University, Uppsala, Sweden Published online: 06 Jul 2015.
To cite this article: Cecilia Engmér Berglin, Pernilla Videhult Pierre, Andreas Ekborn, Tobias Bramer, Katarina Edsman, Malou Hultcrantz & Göran Laurell (2015): Local treatment of the inner ear: A study of three different polymers aimed for middle ear administration, Acta Oto-Laryngologica To link to this article: http://dx.doi.org/10.3109/00016489.2015.1058534
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Acta Oto-Laryngologica. 2015; Early Online, 1–10
ORIGINAL ARTICLE
Local treatment of the inner ear: A study of three different polymers aimed for middle ear administration
CECILIA ENGMÉR BERGLIN1,2, PERNILLA VIDEHULT PIERRE1,3, ANDREAS EKBORN1,2, TOBIAS BRAMER4, KATARINA EDSMAN4, MALOU HULTCRANTZ1,2 & GÖRAN LAURELL3 Downloaded by [University of Otago] at 05:50 13 July 2015
1
Department of Clinical Science, Intervention, and Technology, Karolinska Institutet, Stockholm, Sweden, Department of Otorhinolaryngology, Karolinska University Hospital, Stockholm, Sweden, 3Department of Surgical Sciences and 4Department of Pharmacy, Uppsala University, Uppsala, Sweden
2
Abstract Conclusion: A formulation based on sodium hyaluronate (NaHYA) was the most promising candidate vehicle for intra-tympanic drug administration regarding conductive hearing loss, inflammatory reactions, and elimination. Objectives: Recent advances in inner ear research support the idea of using the middle ear cavity for drug administration to target the inner ear. This paper presents rheological and safety assessments of three candidate polymer formulations for intra-tympanic drug administration. Method: The formulations were based on sodium carboxymethyl cellulose (NaCMC), sodium hyaluronate (NaHYA), and poloxamer 407 (POL). Rheological studies were performed with a controlled rate instrument of the couette type. Safety studies were performed in guinea pigs subjected to an intra-tympanic injection of the formulations. Hearing function was explored with ABR before and 1, 2, and 3 weeks after the injection. Elimination of the formulations marked with coal was explored with an endoscopic digital camera 1, 2, and 3 weeks after injection. Middle and inner ear morphology was examined with light microscopy 6 days after injection. Results: The results speak in favor of NaHYA, since it did not cause prolonged hearing threshold elevations. The results of the elimination and morphological investigations support the conclusion of NaHYA being the most promising candidate for intra-tympanic administration.
Keywords: ABR, carboxymethyl cellulose, guinea pig, hyaluronan, intra-tympanic, morphology, poloxamer 407, polymer, targeted drug delivery, toxicity
Introduction Topical application of drugs is used to treat a variety of sites and organs, such as the eye and the skin. Recent advances in inner ear research support the idea of using the middle ear cavity as a reservoir for local drug administration to target cells in inner ear compartments [1]. Destructive treatment of vertigo with gentamicin in Ménière’s disease is today the only established clinical method that employs intra-tympanic drug administration [1]. In the literature, numerous studies can also be found on intra-tympanic injection of glucocorticoids to treat
different inner ear disorders in humans, such as autoimmune inner ear disease, idiopathic sudden sensorineural hearing loss, tinnitus, or Ménière’s disease, but data supporting their efficacy are still weak [1]. There are a number of advantages of middle ear drug delivery over systemic administration. With local drug delivery, unacceptable side-effects and drug interactions in the blood compartment may be avoided. Moreover, the drug uptake to inner ear compartments may be increased, since these compartments can be difficult to reach from the systemic circulation due to different barrier systems.
Correspondence: Professor Göran Laurell, MD, Uppsala University Hospital, ENT Clinic, SE-751 85 Uppsala, Sweden. Tel: +46 18 6115363. Fax: +46 18 558231. E-mail: [email protected]
(Received 9 April 2015; accepted 25 May 2015) ISSN 0001-6489 print/ISSN 1651-2251 online 2015 Informa Healthcare DOI: 10.3109/00016489.2015.1058534
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The round window membrane is considered to be the most important route for a drug to reach the inner ear when administered intra-tympanically [1], although recent data suggest that the oval window can also be of importance [2]. Therefore, a key factor for drug uptake is the contact time between the drug and this/these membrane/membranes [1]. At present, patients subjected to intra-tympanic drug administration are, therefore, often requested to remain still for about half an hour with the injected ear facing upwards. This is inconvenient, especially if both ears need to be treated at the same time. The problem may be circumvented by using a polymer; the inclusion of a polymer in a drug formulation can improve the mucoadhesive [3] and rheological [4] properties of the formulation, resulting in a prolonged residence time at the site of the absorption and, thereby, a facilitated drug uptake. A semisolid polymer formulation can be in the form of e.g. a gel or a polymer solution. A polymer gel usually consists of a polymer network formed by chemical cross-links or physical interactions [5]. Most gels are soft, solid or solid-like materials consisting of at least two components, one of which is a liquid present in abundance [5]. If a polymer network is lacking, the formulation becomes fluid and is usually classified as a polymer solution [5]. The solidity of a polymer formulation is dependent on its viscoelastic properties. The viscosity is a critical factor of an intra-tympanically applied formulation aimed for treatment of inner ear disorders, since it can be expected that a formulation with a low viscosity will not closely stay in contact with the round and/or oval window membranes and, therefore, most probably give a low drug transport to the inner ear. On the other hand, if the viscosity is too high, the formulation might be hard to eliminate from the middle ear and lead to a conductive hearing loss for a prolonged period of time. Little is known about the elimination mechanisms of an intra-tympanically applied polymer formulation, but Eustachian tube mucociliary transport is likely a fundamental mechanism [6]. The inner ear structures are delicate and research has to be undertaken to develop not only effective but also safe local drug delivery systems in search for potential and novel models. The objective of the experimental studies presented in this paper was to explore three different polymer formulations as candidate vehicles for intra-tympanic drug administration. The explored polymers were sodium carboxymethyl cellulose, sodium hyaluronate, and poloxamer 407. These polymers are already used in different pharmaceutical formulations and are considered non-toxic and non-irritant [7]. Sodium carboxymethyl cellulose is a widely employed
viscosity modifier and emulsifier [7]. It is used, e.g., in artificial tears and as a food additive [7]. Sodium hyaluronate is a linear molecule composed of a repeating disaccharide of N-acetyl-D-glucosamine and D-glucuronic acid [8]. It is naturally and ubiquitously occurring in humans and a major component of the extracellular matrix [8]. It is produced by hyaluronan synthase, is degraded by hyaluronaidases, and is immunologically inert [9]. Poloxamer 407 is a polyoxyethylene polyoxypropylene block copolymer used in several different pharmaceutical preparations, for both internal and external use [7]. First, the rheological properties of the polymer formulations were investigated in vitro. Second, the polymer formulations were administered into the middle ear of guinea pigs and their effects on the electrophysiological hearing thresholds and morphology as well as their elimination were investigated. Materials and methods Animals A total of 35 guinea pigs (Duncan-Hartley) of both sexes were purchased from a local breeder and were maintained on a 12:12 h light/dark cycle with unrestricted access to food and water. The care and use of the animals were approved by the local animal care and use committee, Stockholms Norra Djurförsöksetiska Nämnd (N 334/05), according to the Declaration of Helsinki. Chemicals Sodium carboxymethyl cellulose (molecular weight: 100 kDa; Cekol100 000), linear sodium hyaluronate (approximate molecular weight: 4000 kDa; Healon), and poloxamer 407 (average molecular weight: 12 kDa; Lutrol F 127) were generous gifts from Noviant B.V. (Nijmegen, The Netherlands), Pharmacia Upjohn (Uppsala, Sweden), and BASF Aktiengesellschaft (Ludwigshafen, Germany), respectively. Ketamine (Ketalar, 50 mg/mL, Pfizer AB, Täby, Sweden) and xylazin (Rompun vet, 20 mg/mL, Bayer Health Care AG, Leverkusen, Germany) were used for general anesthesia and lidocaine (Xylocain, 20 mg/mL, AstraZeneca, Södertälje, Sweden) for local anesthesia. Preparation of experimental polymer formulations Experimental polymer formulations were prepared by mixing commercial polymer products with different solvents. A summary is given in Table I. A sodium carboxymethyl cellulose formulation (NaCMC) was
Three polymers
3
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Table I. Experimental polymer formulations were prepared by mixing commercial polymer products with different solvents. Final concentration (% w/w)
Abbreviation of experimental polymer formulation
0.5
NaCMC
~7.2
0.5
NaHYA
~7.2
25
POL
Chemical name
Brand name
Solvent
Final pH
Sodium carboxy-methyl cellulose
Cekol
Saline
7.3–7.5
Sodium hyaluronate
Healon
Phosphate-buffered saline
Poloxamer 407
Lutrol
Phosphate-buffered saline
prepared by dispersing Cekol in saline (9 mg/ml) and allowing it to completely dissolve before pH adjustment with sodium hydroxide. Additional saline was then added to a final polymer concentration of 0.5% (w/w). A sodium hyaluronate formulation (NaHYA) was prepared by mixing Healon (1% w/v) with phosphate-buffered saline to a final polymer concentration of 0.5% (w/w). A poloxamer 407 formulation (POL) was prepared by dissolving Lutrol in phosphate-buffered saline to a final poloxamer 407 concentration of 25% (w/w). For semiquantification of the elimination of the formulations, NaCMC, NaHYA, and POL, respectively, were mixed (5:95, v/v) with a coal suspension (a kind gift from the Karolinska Pharmacy, Stockholm, Sweden). All polymer formulations were autoclaved before being administered to the animals.
subjected to an injection of NaCMC, NaHYA, or POL (volume = 0.15 mL; needle: BD Microlance 30G, external diameter = 0.3 mm) into the auditory bulla through the skin of the auricle after paracentesis of the tympanic membrane. An operating microscope was used to determine the point of injection and to observe the flow of the polymer formulation into the middle ear cavity. The animals were allowed to regain consciousness prior to being returned to the vivarium. The effects and safety of the intra-tympanically administered polymer formulations were studied by hearing assessment using acoustically evoked ABR, by visual semi-quantification of elimination of the polymer formulations marked with coal after opening the auditory bulla, and by light microscopic examination of the middle and inner ears. Acoustically evoked ABR assessment
In vitro experiments NaCMC, NaHYA, and POL were investigated rheologically with a Bohlin VOR Rheometer (Bohlin Reologi, Lund, Sweden), a controlled rate instrument of the couette type. Viscosimetric measurements were carried out on NaCMC and NaHYA at 37 C, using a concentric cylinder system (C8). Oscillating measurements were performed on POL with the same system at 20, 25, 30, 35, and 40 C. The rheological properties of the coal-marked formulations were not examined. In vivo experiments The animals were ~10 weeks old at the start of the experiment. They were anesthetized with ketamine (40 mg/kg b.w., i.m.) and xylazine (12 mg/kg b.w., i.m.). Additional doses of ketamine and xylazine were administered when needed to maintain an adequate depth of anesthesia. The ear canals, tympanic membranes, and electrophysiological hearing thresholds of the animals were verified as being normal by otoscopic examination and by assessment of acoustically evoked auditory brainstem response (ABR; for details, see below). While still under anesthesia, the animals were
Fifteen albino guinea pigs (mean weight = 303 g; weight range = 269–345 g) were used to evaluate the effects of the polymer formulations on acoustically evoked ABR thresholds. The measurements were performed with a TDT system II (Tucker Davies Technologies, Gainesville, FL), a standard modular system operated via a personal computer, using TDT BIO SIG ver. 2.0 software. The sound stimuli were delivered inside the ear canal through a speculum from a TDT high frequency transducer with the animal placed in a soundproof box. The auditory stimuli consisted of 2 ms full sine wave tone bleeps at 3, 6, 12, 20, and 30 kHz designed with a cosine gate and were delivered to the ear at a rate of 30/s. The sound stimulus intensity was varied in 5 dB intervals. The electric signals were collected by stainless steel electrodes placed subcutaneously in the infraauricular region of the measured ear and in the vertex. The reference electrode was placed on the nasal bridge. The signal was amplified and sampled over a 10 ms period, digitally averaged over 2000 cycles, low-pass altered, and presented as an electroencephalogram on the computer screen. The electrophysiological hearing threshold was defined as the lowest intensity at which a reproducible response could be recorded.
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The 15 animals were randomized to be administered NaCMC, NaHYA, or POL into the middle ear cavity bilaterally (n = 5 in each group). ABR assessments were repeated weekly for 3 weeks after the administration. Thresholds were considered elevated if being at least 10 dB higher compared to the baseline value at more than one frequency. After the final ABR assessment, the animals were decapitated while still under deep anesthesia.
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Visual semi-quantification of coal-marked polymer formulation by opening the bulla Nine albino guinea pigs (mean weight = 279 g; weight range = 220–320 g) were used to evaluate the elimination of NaCMC, NaHYA, or POL marked with coal. The animals were randomized into three groups (n = 3 in each group). Each ear of the animals in the different groups was randomized to be administered NaCMC, NaHYA, or POL marked with coal in such a way that no animal received the same polymer formulation in both ears. One animal in each group was re-anesthetized 1, 2, or 3 weeks after the administration. The auditory bullae were opened with a dorsolateral approach. All ears were examined for remnants of the coal-marked polymer formulations and inflammatory response in the middle ear, using a dissecting microscope. Quantification was performed with a four-graded scale (Table II). The middle ears were photographed using an endoscopic digital camera. Finally, the animals were euthanized as described above. Table II. Guinea pigs were subjected to an intra-tympanic injection (0.15 mL) of a polymer formulation of sodium carboxymethyl cellulose (0.5% w/w; NaCMC), sodium hyaluronate (0.5% w/w; NaHYA), or poloxamer 407 (25% w/w; POL) marked with coal. The auditory bullae were opened 1, 2, or 3 weeks after the injection. The signs of remnants of the coal-marked formulations and inflammatory response in the middle ear was quantified using a four-graded scale. Stage
Criteria
A
No visible signs of NaCMC, NaHYA, POL, or coal; no visible signs of inflammation.
B
Visible coal remnants in the middle ear mucosa; no visible signs of NaCMC, NaHYA, POL, or liquid in the middle ear cavity, around the ossicules, or at the round window; no edema in the middle ear mucosa.
C
Visible coal remnants around the ossicules and/or at the round window; edema in or reddened middle ear mucosa or liquid in the middle ear cavity.
D
Middle ear filled with NaCMC, NaHYA, or POL marked with coal.
Light microscopic examination of the middle and inner ears Eleven albino guinea pigs (mean weight = 284 g; weight range = 242–327 g) were used for light microscopic evaluation of inflammatory reactions in the tympanic membrane, the middle ear mucosa, and the round window membrane after intra-tympanic administration of NaCMC, NaHYA, or POL. Light microscopic examination of the hair cells and stria vascularis of the basal turn of the cochlea, close to the round window membrane, was also performed. The animals were randomized to be administered NaCMC, NaHYA, or POL into the middle ear bilaterally (n = 3 in each group). One animal was injected with saline (9 mg/ml) and one received no injection, serving as a control. Six days after the administration, the animals were re-anesthetized and euthanized by decapitation. The auditory bullae were disassembled from the temporal bone and fixed in paraformaldehyde (4%) for 2 h. They were stored in paraformaldehyde (0.5%) at 4 C until decalcified with EDTA (0.1 M in phosphate buffer (0.1 M)), washed in phosphate buffer (0.1 M) several times, dehydrated in a graded ethanol series, and embedded in JB4 resin (Polyscience). Then, they were sectioned at 3-mm thickness with a rotary microtome (Microm HM 355 S). The left ears were sectioned perpendicularly to the RMW and across the cochlea. The right ears were sectioned perpendicularly both to the round window membrane and the tympanic membrane across the middle ear and the cochlea. Every 10th section was mounted on glass slides, stained with toluidine blue (0.1%), and examined under a light microscope (Zeiss) equipped with a digital camera (Altra 20 soft imaging system, Olympus). The thickness of the round window membrane and the tympanic membrane was measured using image analysis software (Cell, Olympus). Measurement of three adjacent sections of each round window membrane and tympanic membrane was made at the mid-portion of the round window membrane and tympanic membrane, respectively, and the value of the thickest part was chosen as a representative value of inflammatory reaction. Data analysis The Wilcoxon signed-rank test was used to compare the median electrophysiological hearing thresholds obtained before the injection of a specific polymer formulation with those obtained after the injection. Kruskal-Wallis and Dunn’s multiple comparison tests were used to compare the hearing thresholds of independent groups. A p-value < 0.05 was
Three polymers considered statistically significant. All statistical tests were two-sided. When the results of an ABR measurement showed that the mean electrophysiological hearing threshold difference of the different frequencies between the two ears of one animal was more than 20 dB, the ear with the lowest threshold was excluded to reduce the risk of erroneous hearing thresholds due to bone conduction of the sound stimuli to the contralateral ear. Results
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In vitro experiments The rheological investigation showed that NaCMC and NaHYA were high viscosity solutions rather than gels (Figure 1). At the shear rates studied, NaCMC exhibited a shear thinning behavior, whereas NaHYA did not (Figure 1). POL formed a semi-solid gel and its rheology changed drastically at a temperature of ~15 C (Figure 2). POL fulfilled a rheological definition of a gel, which is having a frequency independent elastic (G’) modulus and a much higher elastic than viscous (G’’) modulus over a large frequency range (Figure 3) [5,10]. In vivo experiments
5
NaCMC injection, in four animals 2 weeks after NaCMC injection, and in three animals 3 weeks after NaCMC injection. In the NaHYA group, the corresponding figures were two animals 1 week after NaHYA injection, one animal 2 weeks after NaHYA injection, and one animal 3 weeks after NaHYA injection. In the POL group, the ear with the lowest mean threshold was excluded in one animal 3 weeks after POL injection. Ears injected with NaHYA had significantly lower threshold shifts at all evaluated frequencies 1 week after the administration compared to ears injected with POL or NaCMC, at 20 and 30 kHz 2 weeks after the administration compared to ears injected with POL, at 30 kHz 2 weeks after the administration compared to ears injected with NaCMC, and at 12 and 30 kHz 3 weeks after the administration compared to ears injected with NaCMC. There were no significant differences between NaCMCand POL-injected ears.
Visual semi-quantification of coal-marked polymer formulation by opening the bulla The results of the visual semi-quantification of elimination of NaCMC, NaHYA, and POL marked with coal are shown in Table III. As can be seen, in no single ear was there a total elimination of coal. Figure 5
Acoustically evoked ABR assessment. The results of the acoustically evoked ABR assessments are illustrated in Figure 4. Due to an intra-individual side difference in mean electrophysiological hearing threshold exceeding 20 dB, the ear with the lowest mean threshold was excluded in one animal 1 week after
Figure 1. Formulations of sodium carboxymethyl cellulose (0.5% w/w; NaCMC) and sodium hyaluronate (0.5% w/w; NaHYA) were prepared and investigated rheologically. The results of the viscosimetric measurements for NaCMC (closed triangles) and NaHYA (closed squares) at 37 C using a concentric cylinder system (C8) are shown.
Figure 2. A formulation of poloxamer 407 (25% w/w; POL) was prepared and investigated rheologically at 10, 15, 20, 25, 30, 35, and 40 C, using oscillating measurements. The viscoelastic properties of POL at 1 Hz are shown. G¢ and G¢¢ signify the elastic and viscous moduli, respectively.
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Figure 3. The elastic (G’; closed circles) and viscous (G’’; open circles) moduli of POL from an oscillating measurement at 30 C.
shows photographs from a middle ear of stage B (5A and 5B) and stage C (5C and 5D). Light microscopic examination of the middle and inner ears The results of the light microscopic examination of morphological signs of inflammation in the tympanic membrane and the round window membrane are shown in Table IV. Due to technical problems, the number of observations differed between groups. The thickness of the round window membrane in normal, unexposed control ears was close to 10 mm, which is in agreement with previous results [11]. Remnants of the polymer formulation and a swollen mucosa could be seen in the middle ear of animals injected with NaCMC (Figures 6A and B). The mucosa was also swollen in POL-injected ears, but no remnants of the
polymer formulation could be seen. In ears injected with NaHYA, there were neither any signs of inflammation in the mucosa nor any remnants of the polymer formulation (Figure 6C). The hair cells and the stria vascularis of the cochlea, close to the round window membrane, could be evaluated in three ears in the NaHYA group and in two ears in the POL and NaCMC groups, respectively. There were no signs of damage in any specimen. Discussion This paper presents rheological and safety assessments of three different polymer formulations aimed for intra-tympanic drug delivery. The results speak in favor of NaHYA. Especially, in contrast to NaCMC and POL, NaHYA did not cause prolonged hearing
Figure 4. Guinea pigs (n = 15) were subjected to an intra-tympanic injection (0.15 mL) of NaCMC (A), NaHYA (B), or POL (C) bilaterally (n = 5 in each group). Acoustically evoked auditory brainstem response (ABR) assessment was performed before as well as 1, 2, and 3 weeks after the injection. The graphs show the electrophysiological hearing threshold shifts, i.e. the difference between the hearing thresholds obtained before and after (1 week, open circles; 2 weeks, closed circles; 3 weeks; open triangles) the injection. Data are expressed as mean ± SEM. The measured frequencies, 3, 6, 12, 20, and 30 kHz are center frequencies of the presented auditory stimuli.
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Three polymers
Table III. Guinea pigs (n = 9) were subjected to an intra-tympanic injection (0.15 mL) of NaCMC (n = 6 ears), NaHYA (n = 6 ears), or POL (n = 6 ears) marked with coal. Their auditory bullae were opened 1, 2, or 3 weeks after the injection and the middle ears were photographed with an endoscope and digital camera. The photographs were used for semi-quantification of the elimination of the coal-marked polymer formulations. Details of the different stages are given in Table II. Absolute numbers of ears are given. Week 1
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Stage
Week 2
Week 3
NaCMC
NaHYA
POL
NaCMC
NaHYA
POL
NaCMC
NaHYA
POL
A
0
0
0
0
0
0
0
0
0
B
0
1
1
0
0
0
0
2
1
C
2
1
1
2
2
2
2
0
1
D
0
0
0
0
0
0
0
0
0
threshold elevations. The results of the elimination and morphological investigations support the conclusion of NaHYA being the most promising candidate for intra-tympanic administration.
Figure 5. Guinea pigs (n = 9) were subjected to an intra-tympanic injection (0.15 mL) of NaCMC (n = 6 ears), NaHYA (n = 6 ears), or POL (n = 6 ears) marked with coal. Photographs of a middle ear injected with coal-marked NaHYA (A and B) and NaCMC (C and D) 3 weeks earlier are shown. (A) A minor area of coal remnant can be seen through the tympanic membrane to the right of the handle of the malleus. (B) The middle ear cavity from a dorso lateral view after the bulla has been opened. Coal remnants can be seen in the mucosa on the inside of the tympanic membrane (TM) and at the round window niche (arrow). There are no visible signs of inflammation or remnants of the formulation in the middle ear. The open black circle indicates the apex of the cochea. (C). The tympanic membrane is thickened and with liquid in the middle ear. (D) The middle ear cavity from a dorso lateral view after the bulla has been opened. Coal remnants can be seen in the mucosa, especially on the tympanic membrane (TM) and at the round window niche (arrow). Coal-marked formulation surrounds the ossicules between the tympanic membrane and the cochlea. The open black circle indicates the apex of the cochlea.
Electrophysiological examination of the hearing using acoustically evoked ABR is a very common method in experimental hearing research. When the sound stimuli is delivered through the ear canal, as in the present study, the hearing thresholds are expected to be elevated when something resides in the middle ear, obstructing the transmission of sound, i.e. causing a conductive hearing loss. Elevated thresholds can also be due to induced inflammatory reactions causing e.g. changes to the tympanic membrane and toxic effects to the inner ear. The fact that there were no significant threshold shifts in the ears injected with NaHYA implies that most of NaHYA had been eliminated from the middle ear already 1 week after the injection, that its induced inflammatory reactions were reversible, and that NaHYA was not ototoxic. Coal in the middle ear was easily visualized using an endoscopic digital camera. However, our interpretation of the results with the coal-marked polymer formulations is only tentative. It has to be pointed out that coal is not soluble in NaCMC, NaHYA, or POL. Therefore, the fact that in no single ear was there a total elimination of coal remnants does not prove that remnants of NaCMC, NaHYA, or POL were still present in the middle ear. In a previous experimental study on a non-soluble form of a drug in a poloxamer 407-based gel, it was observed that drug microparticulates were present in the middle ear beyond the disappearance of the gel matrix [12]. Furthermore, in the present investigation, the number of observations was few in the coal experiments, which makes it impossible to draw any firm conclusions from this part of the study. However, the results point in the same direction as the results of the ABR assessments. For example, when coal remnants were found around the middle ear ossicles or liquid in the middle ear cavity was apparent (the criteria of Stage C), as in all NaCMC-administered ears 1, 2, and 3 weeks after the administration, effects on the hearing function can be expected, which were also the case in the majority of the NaCMC-administered ears.
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Table IV. The thickness (mm) of the round window membrane and tympanic membrane obtained from guinea pigs 6 days after a bilateral intra-tympanic injection (0.15 mL) of NaCMC (n = 3 ears), NaHYA (n = 3 ears), POL (n = 3 ears) or saline (9 mg/ml; n = 1 ear). Each value represents one sample. NaCMC
NaHYA
POL
Saline
No treatment
Round window Tympanic Round window Tympanic Round window Tympanic Round window Tympanic Round window Tympanic 26
45
14.2
18.2
20.8
54.9
19.6
24.1
13.3
17.9
23.1
54.2
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18.2
The results of the coal experiments support the ABR findings as well as the light microscopy investigation, indicating that NaHYA is the best of the three studied polymer formulations for intratympanic administration. Sodium hyaluronate, which was used for preparation of NaHYA, is the sodium salt of hyaluronic acid. In the literature, hyaluronan is a general term for this polymer, irrespective of its degree of dissociation. Locally applied exogenous hyaluronan has been used for a long time in otological research, both in experimental animals [11,13,14] and humans [15,16]. These studies have shown that it is welltolerated and not ototoxic, even when administered into the middle ear when there has been a direct access to the inner ear [13,14]. The light microscopic investigation described in this paper showed that NaHYA induced less thickening of the round window membrane and tympanic membrane than NaCMC and POL. The effect of NaHYA on the round window membrane agrees with that of a previous study, in which a similar hyaluronan formulation was used [11]. In that study, the thickness of the round window
22.6
30.5
10.1
13.2
10
45.4
membrane had returned to normal values after 4 weeks. Exogenous hyaluronan is eliminated primarily via the Eustachian tube [6], which is a general mechanism fundamental for elimination of products from the middle ear cavity. It may be totally eliminated within a day in the rat, according to an experimental study using a formulation with a hyaluronan concentration twice that of the present investigation [6]. The results of our study using NaHYA marked with coal suggest that a hyaluronan-based formulation may stay in the middle ear considerably longer in the guinea pig. However, there were no statistically significant hearing threshold shifts 1, 2, and 3 weeks after administration of NaHYA. Neither were there any remnants of NaHYA found in the light microscopic investigation 6 days after its administration. Obviously, further studies are needed if the elimination kinetics of NaHYA from the middle ear is to be established. Diverging data on the elimination of exogenous hyaluronan from the middle ear cavity may be due to differences in transport mechanisms in different species, administered volume, and concentration as well as molecular weight of hyaluronan.
Figure 6. Guinea pigs (n = 11) were subjected to a bilateral intra-tympanic injection of NaCMC (n = 3), NaHYA (n = 3), POL (n = 3), saline (n = 1), or no treatment (n = 1). After 6 days, the animals were euthanized for histological examination of the middle and inner ears using light microscopy. A NaCMC-treated (A and B) and a NaHYA-treated (C) middle ear is shown. (A) A submucosal edema can be observed in proximity to remnants of the formulation (arrow) in the middle ear cavity (ME). (B) The round window membrane (RWM) is thickened and the polymer formulation can be seen between the round window membrane and the stapes (bone in top of the picture) in the middle ear cavity (ME). (C) A NaHYA-treated ear sectioned perpendicularly both to the round window membrane (RWM) and the tympanic membrane (TM) across the middle ear cavity (ME) and the cochlea. The round window membrane and the tympanic membrane are slightly thickened, but the mucosa of the middle ear shows no sign of inflammatory reaction. The organ of Corti (OoC) and stria vascularis (SV) in the basal part of the cochlea appear normal.
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Three polymers In the present study, the hyaluronan concentration was not varied, but it is well known that its viscosity is directly proportional to its concentration [8]. As a result, the elimination rate of a hyaluronan formulation from the middle ear cavity will most likely be reduced when the concentration of hyaluronan is increased. Similarly, increasing the molecular weight of hyaluronan will increase the viscosity of the formulation [8]. The molecular weight of hyaluronan depends on the hyaluronan source and on the isolation and purification procedures used [8]. In the present study, all formulations were autoclaved prior to being used in vivo. Hyaluronan has been shown to be sensitive to high temperatures [8]. Bothner Wik [17] has suggested how the average molecular weight (Mw) of hyaluronan can be calculated from a known zero shear viscosity (h0) and concentration (c) [17]. Equation (1) is valid when h0 < 0.01 Pa s, whereas equation (2) is valid when h0 > 0.1 Pa s.
h0 = 1.7 × 10−7 × (c × Mw )
0.74
h0 = 5.3 × 10−26 × (c × Mw )
3.6
(1)
(2 )
Unfortunately, h0 of the hyaluronan formulation used in this study was determined to 0.085 (Figure 1), which means that it lies in a region where the average molecular weight is difficult to calculate using either of these two equations. However, using equation (2) allows us to at least estimate the average molecular weight to ~1000 kDa, indicating a massive degradation of NaHYA following heat sterilization. As illustrated in Figure 1, NaCMC was pseudoplastic, which means that the carboxymethyl cellulose molecules deformed and aligned in the streamlines of flow, resulting in a decrease in the viscosity. This behavior occurs when pushing a pseudoplastic formulation through medical needles, which makes the needles easy to handle. In contrast, NaHYA did not exhibit a shear thinning behavior (Figure 1). Nonetheless, hyaluronan has been shown to be pseudoplastic when applying a higher shear force or when it is less concentrated [8]. Even though sodium carboxymethyl cellulose is considered non-toxic and non-irritant, there are few published studies on the use of sodium carboxymethyl cellulose in otological research, in contrast to hyaluronan. The results of the present study show that NaCMC might be less convenient for intra-tympanic administration than NaHYA. In fact, sodium carboxymethyl cellulose might even be ototoxic, according to a guinea pig study [18]. However, in that study, a commercial
9
formulation aimed for nasal application was used and it is unknown whether the hearing loss was caused by sodium carboxymethyl cellulose itself or by some other component or property of the formulation. Poloxamers are temperature-responsive, being less viscous when refrigerated or at room temperature than at body temperature. To facilitate the intratympanic injection in the present study, samples of POL were taken from the refrigerator immediately prior to the injection. Yet, the viscosity of POL was still high and, to avoid excess trauma to the tympanic membrane, POL as well as NaCMC and NaHYA were injected through the auditory bulla. Similar to sodium carboxymethyl cellulose and hyaluronan, poloxamer 407 is considered non-toxic and nonirritant [7]. Nonetheless, the results of the present study imply that POL is less suitable for intratympanic administration than NaHYA. In particular, POL induced a striking thickening of the tympanic membrane and substantial effects on the hearing thresholds. However, according to previous publications, poloxamer 407 may be well-tolerated also when administered into the middle ear [12,19]. In a study, most of the conductive hearing loss had disappeared ~4 weeks after intra-tympanic administration of poloxamer 407 [12], and several weeks earlier in another study [19]. In these studies, a lower concentration of poloxamer 407 and a smaller injected volume were used, which might explain their more promising results. A variety of delivery systems has been investigated in order to achieve a reproducible and prolonged drug exposure of the inner ear. These include repeated transtympanic injections, instillation through ventilation tubes, and continuous infusion through microcathethers. The method of a single transtympanic injection of a polymer formulation involves no surgery and only minor trauma. Its major drawback is that it induces a transient conductive hearing loss and, thereby, it may not be tolerated by humans. Several experimental animal studies have explored hyaluronan formulations as a vehicle for intra-tympanic administration of pharmacological compounds [11,20]. There are even some clinical studies, in which dexamethasone was administered into the middle ear in a hyaluronan-based formulation [15,16]. The study presented in this paper supports further investigation of hyaluronan for middle ear administration. It also suggests cautiousness regarding middle ear administration of sodium carboxymethyl cellulose, especially since it has been proposed to be ototoxic [18]. Finally, the results using poloxamer 407 were not promising, but may have been due to a too high concentration, since other researchers have
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shown good results when using less concentrated poloxamer 407-based formulations.
Acknowledgments
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The authors acknowledge Staffan Eksborg for the statistical analyses and AFA, the Acta OtoLaryngologica Foundation, and Rosa and Emanuel Nachmansson’s Foundation for their generous financial support for the conduct of the research and preparation of the article. The foundations had no involvement in the study design, in the collection, analysis, and interpretation of data, in the writing of the report nor in the decision to submit the paper for publication. Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
[8]
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