Journal Pre-proofs

In Situ Gelling System for Sustained Intraarticular Delivery of Bupivacaine

and Ketorolac in Sheep

Hani Abdeltawab, Scott M. Bolam, Jagdish K. Jaiswal, Sue R. McGlashan,

Simon W Young, Andrew Hill, Darren Svirskis, Manisha Sharma

PII: S0939-6411(22)00060-1
DOI: https://doi.org/10.1016/j.ejpb.2022.03.012
Reference: EJPB 13748

To appear in: European Journal of Pharmaceutics and Biophar-

maceutics

Received Date: 23 January 2022

Revised Date: 23 March 2022
Accepted Date: 26 March 2022

Please cite this article as: H. Abdeltawab, S.M. Bolam, J.K. Jaiswal, S.R. McGlashan, S.W. Young, A. Hill, D.
Svirskis, M. Sharma, In Situ Gelling System for Sustained Intraarticular Delivery of Bupivacaine and Ketorolac
in Sheep, European Journal of Pharmaceutics and Biopharmaceutics (2022), doi: https://doi.org/10.1016/j.ejpb.
2022.03.012

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Original article
In Situ Gelling System for Sustained Intraarticular Delivery of Bupivacaine and Ketorolac in
Sheep

Hani Abdeltawaba, Scott M. Bolamb,c, Jagdish K. Jaiswald, Sue R McGlashane, Simon W Youngb,f,
Andrew Hillg, Darren Svirskisa, Manisha Sharmaa*

a School of Pharmacy, Faculty of Medical & Health Sciences, The University of Auckland, Auckland,
New Zealand
b Department of Surgery, School of Medicine, Faculty of Medical & Health Sciences, University of
Auckland, Auckland, New Zealand.
c Department of Orthopaedics, Auckland City Hospital, Auckland, New Zealand
d Auckland Cancer Society Research Centre, Faculty of Medical & Health Sciences, and Maurice
Wilkins Centre for Molecular Biodiscovery, The University of Auckland, Auckland, New Zealand.
e Department of Anatomy, Faculty of Medical & Health Sciences, The University of Auckland,
Auckland, New Zealand.
f Department of Orthopaedics, North Shore Hospital, Auckland, New Zealand
g Department of Surgery, School of Medicine, Faculty of Medical & Health Sciences, The University of
Auckland, Middlemore Hospital, Auckland, New Zealand

* Correspondence: Manisha Sharma

Tel.: + 64 9 373 7599 Ext 81830
Fax: +64 9 367 7192
E-mail address: manisha.sharma@auckland.ac.nz
Postal address: School of Pharmacy
Faculty of Medical and Health Sciences
University of Auckland
Private Bag 92019
Auckland, New Zealand
Abstract

Suboptimal control of postoperative pain following knee arthroplasty can slow recovery and
reduce patient satisfaction. Intraarticular (IA) administration of bupivacaine and ketorolac
offers efficient pain control and minimizes opioid consumption. However, the clinical
benefits of this approach are short lived due to rapid clearance of drugs from the joint
cavity. Here, we describe a poloxamer based thermoresponsive in situ gelling system for the
sustained IA delivery of bupivacaine hydrochloride (BH) and ketorolac tromethamine (KT)
following knee surgery in an ovine model. Drug loaded formulations were prepared using
poloxamer 407, poloxamer 188 and sodium chloride. In vitro characterization was
conducted, followed by in vivo evaluation of sustained drug release and safety in an ovine
model of knee joint surgery. Rheological studies revealed a Newtonian-like flow of the
developed formulation at room temperature, confirming its injectability, followed by a
transition to a viscous gel as temperature approached body temperature. The developed
formulation successfully sustained the in vivo release of BH for 72 h and KT for 48 h, as
determined by circulating drug levels, compared to 24 and 8 h for marketed drug solutions.
The concentrations of BH and KT in the synovial fluids at 72 h were 11.5 and 1.8 times that
of marketed products, suggesting a significant increase in the IA residence time. The
developed formulation induced a comparable inflammatory response compared to the
marketed drug solutions, however a significantly higher chondrotoxicity was observed
following administration of the gel formulations. Poloxamers based in situ gelling systems
are promising delivery platforms for the sustained and localised IA delivery of BH and KT,
with potential clinical benefits in managing the postoperative pain following knee
arthroplasty.
Graphical Abstract

Keywords: Arthroplasty; Knee replacement; Postoperative pain; Local delivery; Multimodal

analgesia; Thermoresponsive; Sustained drug release; Poloxamers.

1. Introduction

Total knee arthroplasty is the standard treatment for chronic pain and limited mobility
associated with end-stage knee arthritis [1,2]. However, the postoperative pain following
the surgery remains an unresolved problem [3–5]. Suboptimal control of this pain leads to
patient suffering and dissatisfaction, impaired mobility and rehabilitation, delayed recovery,
prolonged hospitalization and increased financial burdens [3,6–9]. Though opioids are
widely employed to manage arthroplasty associated postoperative pain, they are linked to
respiratory depression, gastrointestinal problems, potential abuse and dependence [10,11].

Intraarticular (IA) administration of local anesthetics (LA), such as bupivacaine hydrochloride

(BH), and non-steroidal anti-inflammatory drugs (NSAIDs), such as ketorolac tromethamine
(KT), are effective in the control of postoperative pain, with limited systemic exposure and
adverse reactions [12–20]. Yet, the clinical benefits of this approach are limited by the short
duration of action due to the rapid clearance of BH and KT from the joint cavity [21,22].
While the continuous infusions of these two drugs can extend the duration of effect, IA
catheters are associated with infection risk and reduced mobility [23]. BH encapsulated
liposomes are reported to provide a sustained drug release [24,25]. However, their IA
administration failed to demonstrate any clinical benefits over a BH solution [26–29]. This is
attributed to the highly porous nature of the synovium, allowing the free movement of the
liposomes between the synovial fluids and systemic circulation [30–32]. Therefore,
developing a delivery platform that addresses the above limitation and offers dual sustained
IA delivery of BH and KT is hypothesized to offer sufficient analgesia and reduce opioid
requirements.

Poloxamer based in situ gelling systems are promising platforms for IA drug delivery [33–
37]. They have been used to deliver diclofenac [38], methotrexate [39], piroxicam [36], and
glucosamine [34] to the joint space. Poloxamers’ concentrated solutions are injectable
liquids at room temperature, allowing IA administration. At physiological temperature, they
form a bioerodible and biocompatible gel, with a potential extension of the release profile
of the loaded drug(s) [40]. We have previously described a poloxamer based formulation for
the dual-sustained release of BH and KT [41]. In that work, the effect of physical blending of
poloxamers 188 and the addition of sodium chloride on the rheological behavior,
mechanical properties, poloxamers supramolecular arrangements and BH/KT release
profiles have been investigated. In addition, the compatibility between BH/KT and the
formulations has been demonstrated [41].

In the present work, we describe an injectable poloxamer based in situ gelling formulation
to provide sustained dual delivery of BH and KT in the joint space in an ovine model of knee
surgery. The formulation composition used in this work is based on our previous in vitro
findings [41], whereas the BH/KT concentrations have been increased based on the findings
of a pilot animal study (data not shown). BH and KT concentrations in blood and synovial
fluid were monitored and analyzed over 72 h. In addition, the safety and toxicity of the
developed formulations were explored by assessing the inflammatory response and cell
viability in synovium and cartilage tissues.

2. Materials and Methods

2.1. Materials

Bupivacaine hydrochloride monohydrate (BH) and ketorolac tromethamine (KT) were

purchased from Jai Radhe Sales (India). Marcaine 0.5%® (bupivacaine hydrochloride 0.5%
w/w) was obtained from AstraZeneca (New Zealand) and Ketolac ampoules® (ketorolac
tromethamine 30 mg/mL) from Amriya Pharmaceuticals (Egypt). Propofol was purchased
from Fresenius Kabi (Germany), isoflurane from MedSource NZ Ltd, (New Zealand),
Cefazolin from AFT Pharmaceuticals Pty Ltd.(Australia), ceftiofur from Zoetis New Zealand
Ltd. (New Zealand), phenobarbital sodium from Provet NZ Pty Ltd., New Zealand, and
Ketoprofen was purchased from Ceva Animal Health (NZ) Ltd. (New Zealand). Antibiotic
Simplex® Bone Cement with Tobramycin was purchased from Stryker Orthopaedics,
Kalamazoo, Michigan (USA). Ketoconazole was purchased from Sigma Aldrich (New
Zealand). Poloxamer 407 (P407), poloxamer 188 (P188) and phosphate-buffered saline (PBS)
tablets were purchased from Sigma-Aldrich (USA). Sodium chloride was purchased from
Sigma Aldrich (UK). Surgical sutures (Vicryl 1, Vicryl 2-0 and Monocryl 3-0 suture were
obtained from Johnson and Johnson Medical Devices, New Brunswick, New Jersey (USA).
Water used in various preparations was obtained by reverse osmosis (0.22 µm Milli-Q,
Millipore, MA) of demineralized water. All other solvents and reagents used were of
analytical grade.

2.2. Preparation of in situ gel formulations

An in situ gelling system was prepared by the cold method, as previously described [40].
Briefly, predetermined weights of P407 (25% w/w), P188 (11% w/w) and NaCl (0.4% w/w)
were dissolved in specified amounts of Milli-Q water by continuous stirring at 4 ℃ until
dissolved. Formulations were loaded with either BH (2.5% w/w) and KT (0.3% w/w) (F1) or
with BH (2.5% w/w) only (F2). The developed formulations were sterilized by autoclaving at
121 ± 3 ℃ for 20 min and stored in the refrigerator (4 ± 2 ℃) protected from UV light.

2.3. Sol-to-gel transition temperature

The sol-to-gel transition temperature of the developed formulations (F1 and F2) was
determined using the tube inversion method, as described by Sharma et al. with
modification [42]. Briefly, formulation (10 mL) was transferred into 15 mL Eppendorf tubes
and placed in a thermomixer (Eppendorf, AG, Germany), previously equilibrated at 20 ℃,
and subjected to temperature increment of 1 ℃ each 1 min till gelation occurred. The
sample was considered gelled if 90° rotation of the Eppendorf tube results in no movement
of the meniscus [42].
2.4. Rheological analysis

The rheological properties were determined using AR-G2 rheometer (TA instruments,
Melbourne, Australia) equipped with a temperature-controlled Peltier plate and a stainless-
steel parallel plate geometry (40 mm). The formulations’ flowability was investigated by
measuring the viscosity as a function of shear rate over the range of 2 - 200 s-1 at 20 ℃.

The viscoelastic behavior was determined at 33 ± 0.1 ℃ (to mimic the knee temperature
[40,43]) by performing an oscillation sweep over an angular frequency range of 0.1 to 100
rad/s within the linear viscoelastic region at a constant and low strain amplitude (0.02 Pa)
[41]. The changes in storage and loss moduli were determined as a function of angular
frequency.

2.5. Injectability

Injectability studies were done using a texture analyzer, attached with a universal syringe rig
(A/USR) attachment (Stable Microsystems, Surrey, UK) [41]. A 3 mL syringe fitted with a 16-
gauge needle was used. The syringe loaded with the formulations was attached to the
syringe rig and the texture analyzer probe, was lowered at a speed of 5 mm/s to compress
the syringe plunger to a distance of 20 mm. The stiction and plateau forces were
determined by using the Exponent software. All experiments were done in triplicate.

2.6. In vitro drug release studies

In vitro drug release studies were conducted in phosphate buffer saline (PBS, pH 7.4) in a
shaking water bath (33 ± 0.5 ℃, 50 rpm) under sink conditions (BH and KT concentrations in
the release medium was never more than 10% of the saturated concentration at 25 ℃ (50
mg/ml [44]) [45]. Briefly, a known amount of sterile formulation (5 g) was placed into a 7 cm
long dialysis bag (SnakeSkin™ Dialysis Tubing, 3.5K MWCO, 35 mm), and the bag was
suspended in a tightly closed glass bottle containing 100 mL of PBS accommodated at 33 ±
0.5 ℃. At predetermined time intervals, aliquots (100 µL) were withdrawn and replaced
with an equal volume of fresh warm medium to maintain sink conditions. The aliquots were
diluted with appropriate volumes of the mobile phase and analyzed using a High-
performance Liquid Chromatography (HPLC) method, as previously described [46]. The
results were expressed as a cumulative percentage drug release against time.
2.7. Animal studies

All applicable international, national and institutional guidelines for the care and use of
animals were followed. All experimentation was approved by the University of Auckland
Animal Ethics Committee (#002123), in accordance with the New Zealand Animal Welfare
Act, 1999. This study conformed to the ARRIVE guidelines [47].

A sheep model was used to study the formulation in vivo performance due to the
comparable body weights and anatomical similarity to the human knee [48]. Twenty-four
Romney ewes aged between 5 and 7 years and weighing 77.6 ± 3.6 kg (mean ± S.D) were
obtained from the Liggin’s Institute Research Farm, The University of Auckland. All the
sheep were examined for general health prior to surgery, and randomly distributed into four
weight-matched groups. The groups’ description and the given treatment are provided in
Table 1. Drug doses were decided based on a pilot study (data not shown) which
demonstrated the dose of the gel groups could be three times the dose in the solution
groups, yet still the peak plasma concentrations remained safe.

Table 1: In vivo study groups, showing the given dose of bupivacaine hydrochloride (BH) and ketorolac
tromethamine (KT).

Study Groups code (treatment) Number Mean weight (kg) ± Drug (dose mg/kg)
of sheep SD
BHKT solution (Marcaine 0.5%® 6 77.1 ± 3.1 BH (2.5) and KT (0.3)
and Ketolac ampoule®)
BHKT gel (Formulation F1) 6 77.8 ± 2.4 BH (7.5) and KT (0.9)
BH solution (Marcaine 0.5%®) 6 77.7 ± 3.7 BH (2.5)
BH gel (Formulation F2) 6 77.7 ± 3.9 BH (7.5)

2.7.1. Surgical procedure

Sheep were fasted for 12 h with free access to water prior to surgery. They were initially
sedated with an intravenous injection of Propofol (2-5 mg/kg). They were then transferred
to an operating room, intubated and maintained under an isoflurane inhalant (1-5%).
Prophylactic antibiotics were given in the form of intravenous Cefazolin (22 mg/kg) and
intramuscular Ceftiofur (2 mg/kg). A permanent catheter was placed into the jugular vein to
administer the antibiotics and to withdraw blood samples. The right hind limb of each sheep
was clipped free of wool to the rump. The surgical site was prepped with betadine/ethanol
and alcohol, then draped sterilely, and prepped a final time with 2% chlorhexidine and 70%
isopropyl alcohol.

As presented in Figure 1, a medial para-patellar incision line was made to access the patella
(the patella tendon remained intact). The patella was shifted laterally to gain access to the
medial femoral condyle (Figure 1A). In order to mimic the trauma associated with knee
arthroplasty, an 8.0 mm diameter hole was drilled in the medial condyle to a 20 mm depth
at an angle approximately parallel to the medullary canal using manual bone reamer drills
(Zimmer Biomet, Warsaw, Indiana USA) (Figure 1B). The drill hole was irrigated with saline.
Antibiotic Simplex® Bone Cement with Tobramycin was prepared as per the manufacturer’s
instructions. Once the cement had developed a sufficient degree of viscosity, it was applied
into the drilled hole and pressurized until the cement finally hardened. Notably, the cement
was placed to a depth such that it did not extend beyond the arc of the condyle (Figure 1C).
The joint space was then thoroughly irrigated with normal saline. The patella was manually
shifted back to the patellar groove, and the leg was worked through its range of motion to
confirm proper articulation.

The joint capsule was then tightly closed with a continuous Vicryl 1 suture, to achieve a
water-tight closure. A 16-gauge needle was then used to inject the allocated treatment
analgesia intraarticularly into the closed joint space at an anteromedial injection site (Figure
1D). The skin was closed with a sub-dermal continuous Vicryl 2-0 suture, then subcuticular
Monocryl 3-0 suture.
Figure 1: Photographs of the surgical procedure and cement plug placement into a medial femoral condyle of a
sheep. (A) Femoral condyle pre-implantation. Arrow indicates the medial condyle where the hole was
surgically drilled. (B) Empty 8.0 mm drill hole following the coring process of the condyle using an 8.0 mm drill
bit. (C) Cement was injected into the drill hole and pressurized. (D) Treatment analgesia was injected
intraarticularly into the closed joint space at an anteromedial injection site.

2.7.2. Postoperative care

Throughout the postoperative period, all sheep were housed indoors in small, individual
holding pens measuring 2.5 m x 2.0 m to limit mobilization. Sheep were recovered and
monitored until they were eating, drinking and standing on their own. They were then
resumed normal weight-bearing activity and were monitored twice daily. At 72 h post-
surgery, all sheep were euthanized with an overdose of phenobarbital sodium (150 mg/kg)
via intravenous injection [49]. Following euthanasia, specimens (synovial membrane and
cartilage) were taken from the limbs (operated and non-operated) and processed for
histological analysis. Distal femurs were dissected of soft tissue and resected, leaving only
the medial and lateral condyle for analysis.

2.7.3. Blood samples collection and determination of BH and KT concentrations

At the specified time points (preoperatively and at 1, 2, 4, 8, 24, 48, 72 h following

treatment administration), blood samples were collected through the catheter placed in the
jugular vein. Catheter was first flushed with saline before blood collection, and the first 5 mL
of blood were discarded to remove heparin from the tube and avoid sample dilution.
Samples were immediately centrifuged (3500 rpm for 10 min at 4 ℃), and the supernatant
(plasma) was removed and stored at – 20 ℃. For drug(s) analysis, the plasma samples were
thawed on ice, an aliquot (20 μL) was withdrawn, and BH/KT were extracted with ice-cold
acetonitrile (4-fold v/v) and analyzed using the developed LC-MS/MS method (see
Supplementary Materials: Tables S1 & S2, and Figures S1 & S2).

2.7.4. Pharmacokinetic analysis

Pharmacokinetic parameters (PK) were calculated using non-compartmental analysis (NCA)

model assuming extravascular input, using a software package PKSolver (a menu-driven
add-in Microsoft Excel, version 2102) [50]. Pharmacokinetics parameters, such as maximum
plasma concentration (Cmax), time to reach the maximum plasma concentration (Tmax),
apparent elimination half-life (T1/2), total area under the plasma concentration-time curve
from zero to time t (AUC 0-t) and from zero to infinity (AUC 0-∞), and the area under the first
moment curve (AUMC), were determined using PKSolver. Dose-normalization was carried
out to compare PK parameters across different doses. The dose-normalized total area under
the plasma concentration-time curve from zero to time t (*AUC 0-t) and from zero to infinity
(*AUC 0-∞), and area under the first moment curve (*AUMC) were calculated by dividing
(AUC 0-t), (AUC 0-∞) and (AUMC) by the drug dose [51,52]. Likewise, the dose-normalized
*Cmax was calculated by dividing Cmax by the drug dose.

2.7.5. Determination of BH and KT concentrations in the synovial fluid

To determine the concentration of BH and KT in the joint cavity at 72 h, the synovial fluid
was collected immediately after euthanasia using a 3 mL syringe, attached to an 18-gauge
needle, and centrifuged at 3500 rpm for 10 min at 4 ℃. The supernatant was collected and
stored at -20 ℃. For analysis, the supernatant was thawed and processed as described
above (section 2.7.3).

2.7.6. Determination of inflammatory response in the synovial membrane

Synovium was collected immediately after euthanasia from treated and non-treated (for
comparison) knees from the patellar, posterior, medial and lateral regions of the knee joint
[53], and directly transferred to a vial containing 10% neutral buffered formalin. For
analysis, the collected synovial membranes were sectionally cut and stained with
Hematoxylin and Eosin (H & E). Images were acquired using a slide scanner optical
microscope (MetaSystems VSlide (brightfield/fluorescence). The obtained images were
scored by two blind observers, semi-quantitively, for three features: hyperplasia of the
synovial lining, the extent of synovial stromal activation and synovial inflammatory
infiltration [54]. Each parameter was scored on a scale from 0 to 3 (0: no inflammation, 1:
mild inflammation, 2: moderate inflammation and 3: severe inflammation). The mean score
was calculated for each animal and rounded to the closest score level (0, 1, 2 and 3).

2.7.7. Cartilage viability studies

Cartilage punches were collected from treated and non-treated knees at the time of
euthanasia, as described in Figure 2, and stained with live-dead dye as described by Li et al.
[55]. Briefly, the samples were incubated overnight at 4 ℃ in an Eppendorf tube containing
1 mL of live-dead dye (composed of Cell Tracker Green - CMFDA (20uM) and Ethidium
homodimer (5uM) in DMEM). On the second day, samples were washed with PBS (three
times, each is for 5 min) and incubated in 4% paraformaldehyde for 1 h. Thereafter, the
samples were washed in PBS (three times, each is for 5 min) and stored in the same solution
at 4 ℃ for imaging. Visualization of the live cells (green fluorescence), and the dead cells
(red fluorescence), were determined at excitation wavelengths of 488 nm or 568 nm, and
the Z-stack images were acquired using a confocal laser scanning microscope (Zeiss LSM 800
Airyscan, manufacturer). Fiji (http://fiji.sc/Fiji, Ashburn, VA, USA) was employed to count
the live-dead cells, as described by Pezzanite et al. [56].
Figure 2: Cartilage sampling sites collected for live-dead staining, showing samples (A) & (B) collected from the
medial condyle, and samples (C) & (D) collected from the lateral condyle.

2.8. Accelerated stability studies

The stability of the in situ gelling formulations was studied for three months in various
storage conditions, namely: 4 ± 2 °C (refrigerator), 25 ± 3 °C / 65% ± 3 humidity (H),
40°C/75% ± 3 H, and 40°C/75% ± 5 H under UV light (light intensity = 3.84 ± 0.05 w/m2)
using Binder stability chambers [57]. At the specified time points (0, 1, 2 and 3 months),
samples were taken and tested for their sol-to-gel transition temperatures, and drug
content using HPLC technique [58].

2.9. Statistical analysis

All results are represented as mean ± standard deviation (SD). The statistical differences
were calculated using one-way analysis of variance (One-way ANOVA) or t-test, which is
applicable [59]. Significance was calculated using GraphPad Prism 8.2.1 (Graph Pad Software
Inc., USA), with a p value of ≤ 0.05 pre-defined as the minimum level of significance.

3. Results and discussion

3.1. Sol-to-gel transition

Determining the sol-to-gel transition temperature of poloxamers-based in situ forming gels

is an indispensable study to ensure their suitability for the intended purposes. As presented
in Table 2, formulations F1 and F2 exhibited a sol-to-gel at 26.3 ℃ and 27.9 ℃, respectively.
The lower sol-to-gel transition temperature demonstrated by F1 could be attributed to the
intermolecular interactions between KT and poloxamers [41,60]. Both formulations
exhibited phase transition at a reasonable time (57 ± 11 and 63 ± 9 s), suggesting their
suitability for the intended purpose. The average temperature of a healthy knee is 33 ℃,
however it might drop to 30 ℃ at surgery time [40,43]. Therefore, the developed
formulations were considered suitable for intraoperative IA administration as they would
remain injectable liquids outside the body, but gel following administration.

Of note, both formulations were transparent colorless solutions, with a pH of 6.02 (F1) and
5.82 (F2), suggesting their suitability for parenteral administration [61]. Our previous
findings demonstrated drug content uniformity with drug loading ranging from 99.13% to
101.7%, which is attributed to the high aqueous solubilities of the BH/KT [44,62].

Table 2: Sol-to-gel transition temperatures and times of F1 and F2 (n = 3), showing the suitability of the
optimized formulations for intraoperative IA administration.
Formulation BH KT Sol-to-gel transition
code (% w/w) (% w/w) Mean ± SD
Temperature (℃) Time (s)
F1 2.5 0.3 26.3 ± 0.3 57 ± 11
F2 25 --- 27.9 ± 0.8 63 ± 9

3.2. Rheological analysis

The rheological properties were investigated at both 20 ℃ and 33 ℃ to identify the flow
behavior at ambient and physiological temperatures. As shown in Figure 3A, both
formulations exhibited Newtonian-like flow at 20 ℃, suggesting their suitability for
injection. As presented, the recorded curves were superimposed, denoting that the
inclusion of KT did not alter the formulation viscosity. At 33 ℃ (Figure 3B), both
formulations demonstrated a significantly higher storage modulus (G’) as compared to the
loss modulus (G’’), independent of the angular frequency, confirming dominant elastic
properties at physiological temperature where the formulations were in the gelled form.

3.3. Injectability

Injectability is a key mechanical property for evaluating formulations intended for

parenteral use [63]. As presented in Table 3, the maximum force required for the
injectability of F1 and F2 was calculated as 1288 and 1083 g, respectively, confirming the
suitability of both formulations for manual injection, as the maximum force required was <
3000 g [64]. Of note, a wide (16-gauge) needle was used in this test, to allow the easy
movement of the viscous formulations. Though this could cause patient discomfort in some
applications, it is not of concern for intraoperative administration, since the patient is under
anesthesia. No significant difference was observed between F1 and F2 in stiction or plateau
forces, as they have the same matrix composition.

Table 3: Injectability (presented as stiction and plateau) forces determined at 20 ℃, showing the suitability of
the developed formulations for the intended purpose (n = 3).
Formulation code Stiction force (g), Mean ± SD Plateau force (g), Mean ± SD
F1 1288 ± 29.5 1194 ± 64.4
F2 1083 ± 66.5 1074 ± 6.7

3.4. In vitro drug release studies

Figure 3C shows the in vitro release profiles of F1 and F2. As presented, both formulations
demonstrated sustained in vitro release of loaded drug(s) over 3 days. This could be
reasonably ascribed to the highly concentrated matrix, reducing the system porosity and
hindering the drug diffusion [41]. Of note, an initial burst release of 10.3% ± 1.4 to 12.4% ±
1.4 (BH) and 13.6% ± 2.7 (KT) was observed within the first hour, respectively.

Of note, F1 and F2 exhibited comparable BH release profiles (similarity coefficient = 82),

denoting that the inclusion of KT did not alter the release behavior of BH [41]. Likewise, the
release profiles of BH and KT were similar (similarity coefficient = 62), which could be
reasonably ascribed to their similar hydrophilic nature and comparable molecular size [41].
A similarity coefficient of 50 or higher indicates the sameness of release profiles [65,66].
Figure 3: Findings of in vitro characterization studies of F1 & F2, showing: (A) Flow properties at 20 ℃, showing
the Newtonian-like flow, (B) Viscoelastic properties, demonstrating a dominant elastic behavior (G') at 33 ℃,
and (C) The in vitro release profiles of BH and KT.

3.5. Animal studies

All surgical procedures were successful with no intraoperative complications. The developed
formulations were easily administered through a needle and exhibited a phase transition in
a reasonable time, and the study ran smoothly until completion. All animals recovered well
in the postoperative period and were able to stand on the operated limb within 1-2 h of
surgery. No animals showed any signs of systemic toxicity (agitation, seizures, respiratory
depression and coma [67,68]). Nonetheless, several sheep in the drug solution groups (BHKT
solution and BH solution) demonstrated postoperative pain and discomfort (weight bearing
on the operated leg, joint limping, holding one leg on the ground, teeth grinding, eating, and
drinking) in the second and third days of surgery, which was managed by intravenous
ketoprofen (1.5 mg/kg). Though the sheep injected with the developed formulations (BHKT
in situ gel and BH in situ gel) did not show any signs of postoperative pain, they were given
ketoprofen for consistency purposes.

3.5.1. Pharmacokinetic analysis

Figure 4 shows the change in mean plasma concentration versus time, whereas selected
pharmacokinetic parameters are presented in Table 4. Despite considerable inter-animal
variations, circulating blood concentrations of both drugs were well below reported toxicity
levels (toxic plasma levels are 2000-3000 ng/mL and ˃ 5000 ng/mL for BH and KT,
respectively) [69–71]. The developed formulations (tested in BH gel and BHKT gel groups)
successfully sustained the BH release for 72 h, as compared to 24 h for the marketed
product (tested in BH solution and BHKT solution groups). Likewise, the BHKT gel group
(injected with F1) demonstrated sustained KT release up to 48 h, compared to 8 h in BHKT
solution group (injected with the marketed products of bupivacaine and ketorolac). The BH
concentration in the synovial fluids after 72 h in BH gel (3150 ± 1270 ng/mL) and BHKT gel
groups (12600 ± 7550 ng/mL) was significantly higher (p < 0.0001, t-test), compared to BH
solution (273 ± 190 ng/mL) and BHKT solution groups (1093 ± 892 ng/mL), respectively. On
the other hand, the synovial KT concentration in the BHKT gel group was 1.8 times that of
BHKT solution group. The findings demonstrated that poloxamers-based in situ forming gels
could significantly extend IA release and residence time of BH and KT, while maintaining
safe systemic levels.

As presented in Figure 4 and Table 4, the developed in situ forming gels exhibited rapid
systemic absorption with an average Tmax of 1.5 h for BH and 2 h for KT, respectively,
compared to a Tmax of 1 h for both drugs in the drug solution groups. However, this initial
bolus release was followed by extended, yet lower, plasma levels for 72 h (BH) and 48 h
(KT). This pattern of drug release could be clinically beneficial for rapid control of the
intense postoperative pain, followed by moderate analgesia over an extended time. Of note,
the longer Tmax observed in the gel groups suggests a delay in the initial drug release, as
compared to the drug solution.

Identifying the maximum drug plasma concentration (Cmax) is crucial in drug development to
assess the systemic safety and release performance of a formulation [72]. As presented in
Table 4, BH and KT showed comparable Cmax between the drug solution (BHKT solution & BH
solution) and the gel (BHKT gel & BH gel) groups. Nonetheless, caution should be exercised,
as the gel groups' dosing was three times the dose given to the drug solution groups. To
exclude the effect of dosing variations, Cmax of each group was dose-normalized (divided by
the given dose) [51,52]. The average normalized *Cmax of BH was calculated as 104.5 ± 17.5
ng/mL and 41.5 ± 12.0 ng/mL for drug solution and gel group, respectively. Likewise, KT
demonstrated a dose-normalized *Cmax of 307.2 ng/mL in BHKT solution group, compared to
118.0 ng/mL in BHKT gel group. The significantly lower (p < 0.0001, t-test) Cmax observed in
the BHKT gel and BH gel groups suggests that the developed in situ gel significantly
retarded/slowed down the in vivo release of BH and KT. This could be attributed to its highly
entangled matrix, hindering the release/diffusion of the loaded drugs. These findings
corroborate with the longer Tmax observed in the gel groups.

The apparent elimination half-life (T1/2) and mean residence time (MRT) are key
pharmacokinetic parameters in understanding the time required for a drug to be eliminated
from the body, and consequently, the pharmacodynamics and duration of action [72,73]. As
shown in Table 4, the gel groups demonstrated significantly higher (p < 0.0001, t-test) T1/2
and MRT for both BH and KT, as compared to the drug solution groups. The mean T1/2 of BH
gel groups was 24.5 ± 3.1 h, compared to 13.6 ± 1.4 h in drug solution groups. Likewise, KT
demonstrated a T1/2 of 11.4 h in BHKT gel group, compared to only 1.9 h in BHKT solution
group. Mean MRT of BH in the gel groups was calculated as 32.6 ± 6.6 h, compared to 17.6 ±
2.1 h in drug solution groups. The MRT of KT in the BHKT gel group was 17.7 h, compared to
2.9 h in the BHKT solution group. The significant extension of BH/KT T1/2 and MRT is
attributed to their slow release rates and longer release profile demonstrated by the gel
groups [73]. Of note, dose-normalized AUC calculations revealed comparable AUC values
among various groups, suggesting a complete release of BH and KT from both dosage forms.

Extending the residence time has been a challenge in the IA drug delivery, attributed to the
highly porous nature of the synovial membrane [21,22]. As shown in Table 4, the
concentration of BH in the synovial fluids in the gel groups at 72 h was 11.5-fold that of the
drug solution groups, denoting that the developed gel formulations successfully extended
the residence time of BH inside the joint cavity for more than three days. On the other hand,
KT concentration in the synovial fluids in the gel group was only 1.8-fold that of the drug
solution group. This extension in BH and KT, IA residence time is likely to offer prolonged
analgesia.

Considerable variations in drug plasma concentrations were observed between animals

within each of the study groups, which was expected [74,75]. These variations could be
attributed to inter-individual variations in sheep pharmacokinetics (drug metabolism and
elimination rate) or the differences in sheep behavior: a higher frequency of sheep
movements and longer-standing time could facilitate the deformation of the gel and
enhance drug release to the systemic circulations. Likewise, increased movement frequency
can also facilitate squeezing the drug solution out of the joint cavity, and therefore, systemic
absorption. However, all the sheep reached to the experimental endpoint with no systemic
toxicity observed.

Though BH gel and BHKT gel groups received three times the dose as compared to drug
solution groups (BH solution and BHKT solution groups, respectively), all the sheep
demonstrated safe plasma levels of both BH (toxic plasma level is 2000-3000 ng/mL [69,70]
and KT (toxic plasma level ˃ 5000 ng/mL, [71]). All the experimental sheep made it to the
experimental endpoint, with no symptoms of systemic toxicity.

Future studies could examine the process of gel degradation in situ. Modern in vivo imaging
approaches could be used to track gel spread and degradation over time. For example, by
adapting approaches used in our previous report where magnetic resonance imaging (MRI)
of a human cadaveric knee was used to track gel following intraarticular injection of a
similar poloxamer formulation [42].

Table 4: Pharmacokinetic parameters following intraarticular administration of 2.5 mg/kg BH and 0.3 mg/kg KT
solutions (BHKT solution group), 7.5 mg/kg BH and 0.9 mg/kg KT in situ gel (BHKT gel group), 2.5 mg/kg BH
solution (BH solution group), and 7.5 mg/kg BH in situ gel (BH gel group). The results suggest that the
developed in situ gelling formulations sustained the drug-plasma release and intraarticular residence time,
compared to the marketed drug solutions. All data are presented as mean (n=6).
PK parameters Drug solution groups In situ gel groups
BH KT BH BH KT BH
BHKT solution group BH solution group BHKT gel group BH gel group
T1/2 (h) 12.6 1.9 14.8 26.8 11.4 22.3
*Cmax (ng/mL) 117 307 92 33 118 50
Tmax (h) 1 1 1 1 2 2
MRT (h) 16 3 19 37 18 28
*AUC 0-t 705 694 560 769 2610 851
(ng/mL*h)
*AUC 0-∞ 932 756 799 910 2774 942
(ng/mL*h)
C48 (ng/mL) ** ** ** 46 7.4 47
C72 (ng/mL) ** ** ** 27 ** 20
Csynovial-72 1093 15 273 12600 28 3150
(ng/mL)
*: Dose-normalized, **: Drug concentration was below the lower limit of quantification.

Figure 4: Drug plasma concentration-time profiles following single intraarticular administration of (A) 2.5
mg/kg BH solution (BHKT solution group) and 7.5 mg/kg BH in situ gel (BHKT gel group), (B) 2.5 mg/kg BH
solution (BH solution group) and 7.5 mg/kg BH in situ gel (BH gel group), and (C) 0.3 mg/kg KT solutions (BHKT
solution group) and 0.9 mg/kg KT gel (BHKT gel group). Each value is the mean (n=6).

3.5.2. Synovial inflammatory response

Representative microscopic images of the synovial membrane are shown in Figure 5. The
data analysis demonstrated that all the study groups exhibited mild synovitis with a mean
score of 0.7 ± 0.3 (BHKT solution), 0.7 ± 0.3 (BHKT gel), 1.3 ± 0.3 (BH solution) and 1.0 ± 0.3
(BH gel). In contrast, the synovium from non-treated knees showed no signs of
inflammation. The inflammatory response observed in the treated knees could be attributed
to the administered drug or secondary to surgical trauma. Notably, BHKT solution and BHKT
gel exhibited slightly lower inflammatory responses, as compared to BH solution and BH gel
groups. This could be attributed to the anti-inflammatory properties of KT. Though the
effect of blank poloxamers on synovial membranes was not assessed in this study, the
findings suggest that poloxamers did not alter the inflammatory response, which is in
agreement with previous reports [76]. The findings suggest the compatibility of the
developed in situ forming gel with synovial membrane.

Figure 5: Representative synovial slides showing mild inflammatory response in all groups from (A)
membranous and (B) fibrous tissue of the synovium. Arrows indicate the type of inflammatory response; blue
arrow: increased thickness of the lining membrane, green arrow: inflammatory infiltration and yellow arrow:
stomal activation.

3.5.3. Cartilage cell viability

Confocal imaging/analysis of chondrocyte viability is a practical approach to quantitively

assess the chondrotoxic activities of intraarticularly administrated pharmaceutical products
[77]. Figures 6 & 7 show the findings of cartilage viability studies. As presented, BHKT and
BH gel groups demonstrated significantly greater chondrocyte death (87% ± 12 and 60% ±
30, respectively) in treated knees, compared to BHKT and BH solution groups (2% ± 2 and
3% ± 2, respectively). Likewise, non-treated knees in the gel groups exhibited greater
chondrocyte death (16% ± 11 and 9% ± 14, respectively), as compared to the non-treated
knee of the drug solution groups (1% ± 1 and 1% ± 0.2, respectively), which could be
attributed to a longer systemic drug-cartilage exposure.

The impact of formulations intended for IA administration on cartilage viability must be

critically evaluated in the preclinical stages of drug development. As presented, the in situ
gelling formulation demonstrated significantly (p < 0.0001, t-test) higher chondrocyte death,
compared to the drug solution groups. This could be attributed to its higher dose, higher
drug concentration and longer drug-cartilage exposure time [78–81]. Several clinical and
laboratory studies have widely reported the chondrotoxicity of LA, including BH [82,83].
Current evidence suggests that the toxicity of LA is dose- and time-dependent [78–82]. Chu
et al. reported that exposure to 0.5% bupivacaine caused significantly higher chondrocyte
death in human and bovine cartilage, compared to 0.25% and 0.125% bupivacaine [84]). The
authors also demonstrated an increase in chondrolysis by increasing the cartilage-
bupivacaine exposure time. Ickert et al. found a significant reduction in viability within 15
min of bupivacaine exposure, and after 60 min of exposure, no vital cells could be detected
in an in vitro study using chondrocytes harvested from arthritic joints [85]. IA pain pump
infusions have been shown to carry the highest risk of chondrolysis and are not advised to
be used around native articular cartilage, for example, in unicompartmental arthroplasty or
soft-tissue reconstruction [82].

Of note, the BHKT gel group demonstrated significantly (p < 0.0001, t-test) higher cartilage
toxicity, compared to BHKT solution group, suggesting chondrotoxic activities of KT when
combined with BH. On the other hand, KT solution (BHKT solution group) did not affect
chondrocyte viability. To the best of our knowledge, current literature lacks any reports on
the impact of intraarticular KT on chondrocyte viability. Yet, a recent in vitro study reported
that incubating the chondrocytes with KT solution (0.3% w/v) for 48 h did not result in a
significant increase in chondrocyte death compared to control [86]. Taken together, it is
suggested that the impact of KT on cartilage viability might be driven by its
dose/concentration and/or time of exposure. Further studies are required to investigate the
impact of sustained release KT formulation on cartilage.

The effect of blank poloxamers-based in situ forming gels on sheep cartilage is not yet
described. However, previous reports demonstrated the compatibility of poloxamers-based
in situ forming gels with other animal models, such as rabbits, rats and minipigs
[34,35,37,38,87]. In addition, it has been reported that P188 significantly reduced in vitro
post-traumatic chondrolysis and enhanced healing of damaged cartilage [88–90].
Nonetheless, further investigations should consider studying the effect of blank
formulations on sheep cartilage.
These findings suggest a potential chondrotoxicity of the developed in situ forming gels in
humans. However, it might not be a major concern in total joint arthroplasty since the
arthritic cartilage is completely resurfaced during the operation.

Figure 6: Bar graph presenting the percentage of dead cells in treated and non-treated knees in study groups,
showing the significantly higher chondrolysis in the gel groups (BHKT and BH). Data are presented as mean ±
SD, where n = 6.

Figure 7: Representative images of live/dead stained cartilage image en face from (A) control, (B) BHKT
solution, (C) BHKT gel, (D) BH solution, and (E) BH gel groups. The green-stained represent viable
chondrocytes, and the red-stained nuclei represent dead chondrocytes. The figure shows that the in situ
gelling formulations (images C and E) triggered greater chondrocyte death rates than drug solutions (images B
& D).

3.6. Accelerated stability studies

Accelerated stability studies were conducted to identify conditions at which the
formulations could maintain their inherent properties and drug content. As presented in
Table 5, no significant (p ˃ 0.05, One-Way ANOVA) changes in sol-to-gel transition
temperatures were observed at 4 ℃, 25 ℃ and 40 ℃ testing conditions over 3 months when
UV light was excluded. On the contrary, a significant (p < 0.05, One-Way ANOVA) increase in
sol-to-gel transition temperatures and solution viscosity as a function of time was recorded
for samples stored under UV light. Interestingly, these changes were also accompanied by a
pH drop from 6.02 to 3.85 (F1) and from 5.82 to 2.66 (F2), which could be attributed to the
degradation of poloxamers into formic and acetic acids [91].

As presented in Figure 8A, BH and KT maintained 100% ± 5.0 of their initial concentrations
when protected from UV light regardless of the temperature, i.e. at 4 ℃, 25 ℃ and 40 ℃.
On the contrary, both drugs exhibited photolysis when subjected to UV light (light intensity
= 3.84 ± 0.05 w/m2) at 40 ℃. Of note, BH exhibited a slower photodegradation rate, with a
total loss of 13.2% ± 3.3 by the end of the study, i.e. three months. On the other hand, KT
lost 100% ± 0.0 of its initial concentration in less than one month, corroborating the
literature [92,93].

Table 5: Changes in the sol-to-gel transition temperature (℃) of formulations F1 and F2 as a function of time at
various testing conditions. Data are presented as mean ± SD, where n =3.
Storage time Formulation F1 Formulation F2
(Months) 4℃ 25 ℃ 40 ℃ 40 ℃/ 4℃ 25 ℃ 40 ℃ 40 ℃/
UV* UV*
0 26 ± 0.3 27.9 ± 0.8
1 26 ± 0.0 26 ± 0.0 27 ± 0.3 31 ± 0.0 27 ± 0.3 27 ± 0.0 28 ± 0.3 36 ± 0.6
2 27 ± 0.0 27 ± 0.0 28 ± 0.6 37 ± 0.6 28 ± 0.6 28 ± 0.3 29 ± 0.6 45 ± 1.0
3 27 ± 0.3 27 ± 0.0 28 ± 0.6 49 ± 0.6 28 ± 0.0 27 ± 0.0 28 ± 0.0 ˃ 50
* 40 ℃/UV: test was carried out at 40 ℃ under UV light (light intensity = 3.84 ± 0.05 w/m2).
Figure 8: Stability studies’ findings showing the stability of BH and KT loaded F1 at various testing conditions
over three months. The results suggest the stability of the developed formulations under various testing
conditions, except of UV light.

4. Conclusion
We have demonstrated the development, in vitro and in vivo evaluation of an injectable
poloxamer based in situ gelling system for the sustained IA delivery of bupivacaine
hydrochloride (BH) and ketorolac tromethamine (KT) following knee surgery. The developed
formulations successfully extended the IA residence time of KT and BH for 48 and 72 h, as
compared to 8 and 24 h for the marketed drug solutions, respectively. Pharmacokinetic
studies demonstrated safe plasma levels of BH and KT. The developed in situ gels did not
result in an increase in synovitis. Chondrotoxicity was observed with the developed
formulations and, therefore, caution should be required, except for total knee arthroplasty,
where cartilage is completely resurfaced. This is the first formulation to offer a
simultaneous, sustained and localized IA release of BH and KT. Further studies are required
to assess the potential clinical benefits of the developed formulations for managing
perioperative pain following knee arthroplasty.

Declaration of competing of interest

The authors declare no conflict of interest.

Acknowledgment

The authors are thankful to Dr. Satya Amirapu and Mrs Jacqueline Ross for their help in
histological analysis, and to Dr. Mark Oliver for help with the animal surgeries.

Funding

This work was funded by the New Zealand Pharmacy Education and Research Foundation
[NZPERF] project grant [No. 3719172, New Zealand] and School of Pharmacy Performance
Based Research Fund [SOP PBRF] [No. 71605, New Zealand].

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Supporting Information
Table S1: Optimized chromatographic conditions of the developed LC-MS/MS method

Time (min) 0.1% Formic acid/water 0.1% Formic acid/acetonitrile Flow rate
(%)* (%)** (mL/min)

0.5 90 10

0.75 10 90

1.75 10 90 0.5

2.5 90 10

4 90 10

*: line A, **: line B

Table S2: Accuracy (calculated as % recovery) and precision (calculated as the coefficient of variation (CV)) of
the developed LC-MS/MS analytical method.

Nominal Calculated conc. (ng/mL) Mean SD Accuracy Precision

conc. calculated
1 2 3 (% recovery) (% CV)
(ng/mL) conc.

Bupivacaine

40 52.2 47.5 42.9 47.5 4.6 118.9 9.7

400 375.2 398.3 348.9 374.1 24.7 93.5 6.6

2000 1778.4 2137.8 2206.1 2040.8 229.7 102.0 11.2

Ketorolac

40 47.0 45.3 46.7 46.3 0.9 115.8 3.2

400 359.0 368.6 354.1 360.5 7.3 90.1 2.0

2000 1890.3 2108.9 2057.4 2018.9 114.2 100.9 5.6

Figure S1: Calibration curves of (A) bupivacaine and (B) ketorolac, showing the acceptable method linearity
over the range 3-3000 ng/mL. Insets present the constructed curves over the concentration range of 3-3000
ng/mL.

Figure S2: Chromatograms of (A) blank sheep plasma, (B) the lower limit of detection and (C) the lower limit of
quantification of bupivacaine and ketorolac, as determined by the signal/noise ratio.