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Abstract
Background Dexamethasone palmitate (DEP), a prodrug of dexamethasone (DEX), is a synthetic corticosteroid medication distinguished by the inclusion of a fatty acid component known as palmitate. This study introduces DEP as a novel therapeutic option for spinal epidural injection, aiming to provide safer and longer-lasting pain relief as an alternative to for patients with spinal stenosis.
Methods 40 rats were randomly divided into four groups: those receiving epidural administration of normal saline (NS), and DEP in the lumbar spinal stenosis (LSS) model, and non-model rats receiving epidural NS administration. Paw withdrawal thresholds to mechanical stimulation and motor function (neurogenic intermittent claudication) were observed for up to 21 days. Hematology and blood chemistry analyses were performed 1 week after drug therapy. Tissue samples were collected for steroid pathology examination to evaluate adhesion degree, perineural area inflammation, and chromatolysis in the dorsal root ganglion (DRG), and adrenal gland.
Results The DEX and DEP groups demonstrated significant recovery from mechanical allodynia and motor dysfunction after 2 weeks of drug therapy (p<0.001). However, by the third week, the effect of DEX started to diminish while the effect of DEP persisted. Furthermore, the DEP group exhibited reduced fibrosis and less chromatolysis than the NS group. No steroid overdose or toxin was observed in any group.
Conclusion The epidural administration of DEP demonstrated therapeutic efficacy in reducing allodynia and hyperalgesia resulting from chronic DRG compression, thus offering prolonged pain relief. These findings underscore the potential of DEP as a promising treatment alternative for pain associated with LSS, serving as a viable substitute for .
- Injections, Spinal
- Back Pain
- Pain Management
- Drug-Related Side Effects and Adverse Reactions
Data availability statement
Data sharing not applicable as no datasets generated and/or analyzed for this study. No data are available.
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WHAT IS ALREADY KNOWN ON THIS TOPIC
Dexamethasone is already used in epidural injections and has demonstrated efficacy for low back pain. However, it comes with certain side effects, which limit its frequency of administration. Dexamethasone palmitate, a prodrug of dexamethasone, may serve as an alternative medication for dexamethasone.
WHAT THIS STUDY ADDS
Dexamethasone palmitate has been used in treatments for conditions such as rheumatoid arthritis and intravitreal injections, among others. However, there has been limited research on its use in epidural injections. This study represents the first investigation into the efficacy of epidural injection of dexamethasone palmitate.
HOW THIS STUDY MIGHT AFFECT RESEARCH, PRACTICE OR POLICY
The epidural administration of dexamethasone palmitate demonstrated therapeutic efficacy in alleviating allodynia and hyperalgesia, offering sustained pain relief. These findings indicate the potential of dexamethasone palmitate as a treatment for lumbar spinal stenosis-related pain.
Introduction
Lumbar spinal stenosis (LSS) is a potentially debilitating condition, often leading to symptoms such as pain, tingling sensation, numbness, muscle fatigue, and leg weakness.1 Due to aging and degenerative changes, LSS affects a significant portion of the population, with prevalence rates ranging from 11% to 39%.2 The management of LSS involves non-surgical and surgical approaches. When non-surgical treatments fail to provide relief, various surgical procedures are employed.3 Non-surgical management options encompass pharmaceutical interventions, physiotherapy, spinal injections, lifestyle modifications, and multidisciplinary rehabilitation programs. While epidural injections of corticosteroids have demonstrated immediate improvements in pain and function, particularly for radiculopathy, their effectiveness in treating spinal stenosis is somewhat limited.4 Therefore, there is a pressing need for a more efficient and safer drug suitable for epidural injections.
Dexamethasone, a synthetic corticosteroid medication, is widely used for its potent anti-inflammatory and immunosuppressant effects in treating various conditions such as allergies, asthma, arthritis, and pain management. In epidural injections, dexamethasone exerts its therapeutic action by targeting key inflammatory mediators, including tumor necrosis factor-alpha (TNF-alpha). By inhibiting the synthesis and release of TNF-alpha, dexamethasone effectively mitigates inflammation, thereby alleviating symptoms like pain and swelling. Moreover, dexamethasone demonstrates broad-spectrum anti-inflammatory activity by suppressing the production of other cytokines like interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and prostaglandins. Dexamethasone palmitate (DEP) serves as a prodrug of dexamethasone, composed of dexamethasone combined with a fat emulsion containing lecithin. The addition of a C16 chain in DEP enhances its hydrophobicity, facilitating strong interactions with phospholipid aliphatic chains through hydrophobic interactions.5 This unique property allows DEP to exhibit high uptake by activated macrophages and ensures a robust distribution capacity in inflammatory tissues while minimizing the risk of adverse effects. Consequently, DEP demonstrates similar anti-inflammatory properties to dexamethasone, effectively reducing levels of inflammatory cytokines such as IL-6, TNF-alpha, and IL-1β, as evidenced by numerous studies.6 7
Recently, dexamethasone has become widely used in epidural injections for pain management. Numerous studies have demonstrated that dexamethasone therapy significantly lowers pain scores and decreases narcotic consumption.8 9 However, it is crucial to acknowledge that epidural steroid injections can be associated with various complications. These may include hormonal change, embolization agents affecting spinal or cerebral blood vessels, potentially leading to ischemic events and strokes due to the size of crystals or particles.10–12 It is noteworthy that non-particulate steroids can form crystals when combined with local anesthetics. Nevertheless, DEP and all local anesthetics tested did not form significant particulates.13 DEP stands as a candidate for a mixture, but further studies on its safety and effectiveness are necessary.
In this study, we hypothesized that epidural administration of DEP could offer numerous advantages in the management of spinal pain. However, no preclinical studies have been conducted to date to assess the safety and efficacy of epidurally injected DEP. In this context, we aimed to explore the potential of epidurally administered DEP, considering it as a therapeutic option for epidural injection.
Methods
Animals
This study adhered to the National Research Council’s guide for laboratory animal care. 40 male Sprague-Dawley rats were individually accommodated within an environment adhering to a 12-hour light/dark cycle for a 1-week acclimation period. During this period, ad libitum access to both food and water was facilitated. To mitigate potential confounding effects of handling on subsequent behavioral responses, all rats underwent a standardized training regimen within the testing environment, conducted a minimum of three times prior to the initiation of experimental procedures. The cohort of rats was randomly divided into four groups: sham group (n=10), normal saline (NS) group (n=10), dexamethasone group (n=10) and DEP group (n=10).
Establishment of spinal stenosis models
Male Sprague-Dawley rats weighing about 250 g were used in this study. The rats were acclimatized for a minimum of 1 week before surgery. In this study, a surgical procedure was performed to create chronic compression on the intravertebral foramen. The procedure involved making an incision on the skin located on the left side of the lumbar vertebrae, between L4 and L6, and separating the left paraspinal muscles from the mammillary and transverse process at the L4–L6 level. The L5 intravertebral foramen was then exposed, and a 3 mm long, stainless-steel rod with a diameter of 0.6 mm was inserted into the foramen. The rod was inserted at a 30° angle relative to the dorsal middle line and 10° relative to the vertebral horizontal line. On reaching the dorsal root ganglia (DRG), there was a brief and slight twitch observed in the hind leg muscles on the operated side.14 For the sham group, the procedure is the same as the experimental group except for rod insertion.
Epidural catheterization
The induction of anesthesia commenced by placing a rat within a confined chamber containing 3% sevoflurane in oxygen (3 L/min) under spontaneous ventilation. Subsequently, 2%–3% sevoflurane was administered via a mask to maintain anesthesia following the onset of unconsciousness, occurring approximately 3 min postinduction. The fur in the target area was trimmed, and the skin was disinfected.
A 2–3 cm incision was made near T13–L1, with the rat positioned to arch its lower back for easier needle insertion. A 20 G sterile intravascular catheter (Kovacs-Cath, Korea) was used to locate the interspinous space between T13 and L1. After confirming entry, the needle was directed inferiorly until engaging the interspinous ligament. While the use of loss of resistance syringes was challenging, the resistance-free transition from the ligament to the epidural space was palpable on careful advancement of the needle. Subsequently, the stylet was removed, and cases were excluded if aspirated blood or cerebrospinal fluid was detected. A 15 cm long polyethylene tubing catheter (PE-10; Natsume Co, Japan) was introduced into the epidural space using aseptic techniques. The catheter was advanced caudally by approximately 3 cm, traversing the space between L4 and L5. The puncture site was sealed, and closure involved suturing the fascia and skin, followed by the application of antibiotic ointment.
Verification included injecting 0.15 mL of 2% lidocaine postrecovery. Correct placement manifested as posterior leg paralysis with maintained anterior leg function. Incorrect placement, indicated by sudden respiratory distress, with or without cardiac arrest, led to exclusion. Rats were observed for 3 days for abnormal posture, vertebral deformity, or behavior, resulting in exclusion for deviations.
Drug preparation
Under general anesthesia (3% sevoflurane in oxygen), epidural administration was performed using isotonic sodium chloride solution (NS) in the LSS model (group NS, n=10); epidural DEP (Lipothason, 4 mg/mL, KIMS, Seoul, Korea) in the LSS model (group DEX, n=10); and epidural dexamethasone (Dexamethasone Disodium Phosphate Injection, 5 mg/mL, Yuhan, Seoul, Korea) in the LSS model (group DEP, n=10). Non-model rats received epidural NS administration (group sham, n=10). A single injection of DEP (0.8 mg/kg), DEX (0.5 mg/kg), or NS (250 µL) was administered. All injections needed to be administered slowly, taking approximately 1 min.
Blood sample collection
After 1 week of drug administration, all rats underwent a blood test using retro-orbital sampling. Rats were subjected to a 12-hour fasting period before the blood appointment. General anesthesia was administered to each rat, and a Pasteur pipette was carefully inserted at the medial canthus under the nictitating membrane at a 45° angle, directed toward the back of the orbit. The plexus was then delicately penetrated with a sterile instrument for blood collection, yielding a total volume of 500 µL.
Separated blood samples were allocated to tubes containing either lithium heparin or trisodium EDTA as anticoagulants. Heparinized samples were centrifuged at 1900×g at 4°C for 10 min, and the resulting plasma supernatants were transferred to fresh tubes for blood biochemistry analysis using a Vetscan vs2 chemical analyzer (Vetscan vs2 Chemistry Analyzer Support Resources, USA). Hematological investigations were conducted using the MASCOT Hematology Analyzer (Drew Scientific Mascot Veterinary Multi-Species Hematology System Hemavet 850 FS, BaneBio).
Behavioral observations
The behavioral tests, such as von Frey Filament test and Treadmill test, employed in this study are comparable to those used in our prior research.15 Briefly, the rats’ behavior was closely monitored for any signs of abnormal posture, lameness, or changes in eating habits. The assessment of motor function and sensory response to mechanical stimuli were used to evaluate the rats’ behavior, with observations made preoperatively and on postoperative days 1, 3, 7, 14 and 21. To ensure unbiased evaluation, a single investigator blinded to the experimental procedure conducted all assessments.
Histopathological examination
All rats were sacrificed for microscopic examination on the completion of behavioral observations. General anesthesia was induced using 3 L/min of oxygen and 2% sevoflurane. Subsequently, transcardial perfusion was carried out on each rat with 200 mL of NS and 300–500 mL of 4% paraformaldehyde in a 0.1 M phosphate buffer solution.
En bloc resection of the vertebral columns from L4 to L6, encompassing the DRG, nerve root, spinal cord, and spinal nerve of L5, was conducted. The overlying muscles were removed to facilitate microscopic examination, focusing on chromatolysis, perineural inflammation, and fibrosis. Stainless steel rods not appropriately inserted into neural foramina were excluded from the test results. Additionally, the quantification of the adrenal gland included each cortical layer (ie, zona reticularis, zona glomerulosa, and zona fasciculata), as well as the adrenal medulla, scale bar=500 µm. The sample underwent postfixation in a 10% buffered formalin solution for 48 hours and decalcification using 10% w/v ethylenediaminetetraacetic acid before embedding in a paraffin block. H&E staining was performed on 4 µm sections, and a microscopic assessment of the tissues was conducted by two pathologists who were blinded to the test group. A pathologist and a deep learning program collaborate to assess chromatolysis in DRG neurons.
Inflammatory and fibrotic degrees were evaluated using methods employed in previous studies: epineural inflammation (grade 0: absence, grade 1: one focus of at least five mononuclear inflammatory cells, grade 2: more than one focus of grade 1 or at least one focus of 5–20 mononuclear inflammatory cells, grade 3: multiple confluent foci of grade 2, grade 4: diffuse and dense inflammation) and epineural fibrosis (grade 0: absence, grade 1: loose and focal (<50%) fibrosis, grade 2: loose and diffuse (>50%) fibrosis, grade 3: dense and focal fibrosis, grade 4: dense and diffuse fibrosis).16 17
Statistical analysis
First, data normality using the Shapiro-Wilk test, confirming that all data were normally distributed (p>0.05). Continuous data are presented as mean±SD. Preoperative values (day 0) and predrug postoperative values (day 6) of the behavioral tests were calculated as the average value of the results obtained over 3 and 2 consecutive days, respectively. Intergroup comparisons during the study period were conducted using the one-way analysis of variance test. In cases where a significant difference was detected, the Bonferroni test was employed as a post hoc comparison between the groups. All statistical analyses were performed by using SPSS (V.20.0; IBM). A p<0.05 was considered statistically significant.
Results
Figure 1 depicts the study timeline and outlines key events. Initially, 40 rats were randomly assigned to four groups, followed by preoperative behavioral tests to evaluate their responses to the environment in the initial days. On the third day, both the foraminal stenosis model and sham surgery were performed. Subsequently, epidural injections were administered via a catheter on the sixth day. After 1 week of drug therapy, hematology and blood biochemistry analyses were performed. Throughout the study, the von Frey test and treadmill test were administered regularly. Finally, on completion of the study, the rats were sacrificed, and pathology examinations were conducted. It is noteworthy that the rats’ weight exhibited a steady increase during the study period. However, 3 days poststeroid injection, a temporary decrease in weight was observed in the respective groups, followed by a subsequent increase in weight (figure 2A).
Timeline of the experimental protocol (created with biorender.com).
(A) Changes in body weight throughout the experiment. Data are presented as the mean±SEM. (B) Withdrawal threshold to the von Frey test on the left paw. Data are presented as the mean±SEM. (C) Withdrawal threshold to the von Frey test on the right paw. Data are presented as the mean±SEM. (D) Distances in a treadmill test. Data are presented as the mean±SEM. aSignificant at p<0.05, DEP group compared with the sham group. bSignificant at p<0.05, DEP compared with the NS group. cSignificant at p<0.05, DEP compared with the DEX group. DEP, dexamethasone palmitate; DEX, dexamethasone; FS, foraminal stenosis; NS, normal saline; SHAM, no model group.
Blood biochemistry and hematology assessment following drug administration
After 1 week of drug administration, the mean levels of hematology and blood biochemistry all fall within the normal range (table 1). In both the DEP and dexamethasone groups, two rats exhibited higher levels of neutrophils (NE%) and lower levels of lymphocytes (LY%), respectively. All other hematology results remained within normal limits. Regarding blood biochemistry results, no significant differences were observed within each group, and all rats exhibited values within the normal range. This suggests that appropriate drug administration did not significantly impact the functions of the liver, kidneys, or glucose metabolism.
Hematological and biochemical parameters of Sprague‐Dawley rats
Mechanical withdrawal threshold
As the surgery was conducted on the left side in all groups, no significant changes were observed in the von Frey test on the right hind paws (figure 2C). For the left hind paws, during the first 3 days, there were no significant differences in the baseline behavior test across all groups. On the sixth day, 3 days after surgery, a decrease from the baseline test was noted. Postsurgery, there was an increasing trend in the sham and DEP groups (figure 2B).
When comparing DEP with the NS group, no significant differences were observed in the first 9 days. On days 13 and 20, the DEP group showed higher values than the NS group (p<0.01), and on day 27, the DEP group exhibited a similar response to the SHAM group (p>0.05). In the initial 20 days, there were no significant differences between the DEP and dexamethasone (DEX) groups. However, on day 27, the DEP group showed signs of recovery from pain while the DEX group exhibited a decrease in recovery (p<0.01).
Motor function test
In the sham group, as the rats grew, the distance they covered on the treadmill also increased. Initially, there were no differences between the groups(p>0.05). However, after surgery, all rats exhibited significantly reduced running distances. In the NS group, the distances remained consistently shorter than the sham group after surgery (p<0.0001).
The DEP group showed a steady increase after drug administration but did not fully recover compared with the sham group on day 27 (p<0.05). The dexamethasone (DEX) group exhibited an increase in the first week after drug therapy, which lasted longer than the DEP group (p<0.01). Subsequently, there was a decreasing trend, and on the final day, the distance covered by the DEP group was longer than the DEX group (p<0.01). When comparing the DEP and NS groups, the DEP group covered significantly longer distances on day 20 (p<0.001) and day 27 (p<0.0001) (figure 2D).
Histological analysis
The proportion of epineurial inflammation in both the DEP and DEX groups was lower than that in the NS group (table 2, figure 3). Similarly, the proportion of epineurial fibrosis in the DEP and DEX groups was also lower than in the NS group (table 3, figure 3). Regarding DRG chromatolysis, which includes both segmental and central chromatolysis, the numbers in the DEP and DEX groups were lower than in the NS group but higher than in the sham group (figure 4A–C), significant p values in the results were corrected. In terms of adrenal gland histology, we assessed the areas of the zona reticularis, zona glomerulosa, and zona fasciculata, along with the adrenal medulla, and found no significant differences between each group (figure 4D). For the spinal cord examination, there was no evidence of central chromatolysis, leptomeningeal inflammation, leptomeningeal fibrosis (synechia), peripheral neuritis, infarct, or degenerative myelopathy in any group. Additionally, there was no vacuolation of the dorsal funiculus in any group. Therefore, at the short term and safe doses of dexamethasone and DEP, no significant histological changes were observed in the spinal cord and adrenal gland. During the assessment of safe drug dosing, rats underwent pathology examinations without any adverse effects observed. This suggests that DEP could be a potential drug for epidural injection.
Histological evaluation of epineurial inflammation in dorsal root ganglion
Histological evaluation of epineurial fibrosis in dorsal root ganglion
Microscopic findings of the left dorsal root ganglia in each group, 21 days after the epidural injection (H&E stain, ×200). (A) Dense and diffuse fibrosis (grade 4) of the epineurium in the NS group. (B) Diffuse and dense infiltration of inflammatory cells (grade 4) in the NS group. (C) Minimal fibrosis (grade 0) of the epineurium in the sham group, in contrast to B. (D) No infiltration of inflammatory cells (grade 0) in the sham group, in contrast to A. (E) Slight fibrosis (grade 0) of the epineurium in the DEP group, in contrast to B. (F) No infiltration of inflammatory cells (grade 1) in the DEP group, in contrast to A. (G) Minimal fibrosis (grade 1) of the epineurium in the DEX group, in contrast to B. (H) No infiltration of inflammatory cells (grade 0) in the DEX group, in contrast to A. DEP, dexamethasone palmitate; DEX, dexamethasone; NS, normal saline.
Microscopic findings of the left dorsal root ganglia and adrenal gland in each group. (A) Percentage of segmental chromatolysis in the left dorsal root ganglion after 21 epidural therapies. (B) Percentage of central chromatolysis in the left dorsal root ganglion after 21 epidural therapies. (C) Overall percentage of chromatolysis in the left dorsal root ganglion after 21 epidural therapies. (D) Area measurements of the adrenal gland after 21 epidural therapies. Significant p values in the results were corrected. *p<0.05, ****p<0.0001. DEP, dexamethasone palmitate; DEX, dexamethasone; FS, foraminal stenosis; NS, normal saline; SHAM, no model group.
Discussion
This study marks the inaugural exploration into the effects and safety considerations associated with epidural administration of DEP concerning mechanical allodynia and motor dysfunction induced by LSS. Our findings indicate a significant enhancement in alleviating mechanical allodynia and neurogenic intermittent claudication in a rat model of LSS through the epidural injection of DEP. Notably, this improvement in pain relief time surpassed that achieved with dexamethasone. Importantly, within the administered safe dose, no histological damage indicative of neurotoxicity was discernible in the DRG, spinal cord, and adrenal gland of experimental rats’ post-DEP injection. These observations underscore the potential therapeutic efficacy of DEP in the context of spinal stenosis treatment. However, there are limitations to consider. The dosage was determined based on equivalence to dexamethasone, which may not be optimal. Further research is needed to explore dose–response relationships. Additionally, the low dose used may have masked potential side effects, and future studies should investigate this further. Lastly, the long-term therapeutic mechanisms of DEP remain unclear, and future research should focus on understanding its effects on mechanical allodynia and motor dysfunction.
DEP has been extensively studied for various medical conditions including rheumatoid arthritis, intercostal nerve block, and hemophagocytic lymphohistiocytosis.6 7 18 In the realm of pain management, it has been explored for musculoskeletal disorders and lumbar facet syndrome.6 19 Previous research has demonstrated the safety and efficacy of DEP, with no reported side effects. However, there is a notable gap in the literature regarding its use in epidural injection, making this study the first of its kind.
DEP emulsion is a liposomal formulation of dexamethasone known for its high lipid solubility and affinity for phagocytic cells. This unique property allows DEP to primarily target inflammatory cells while minimizing exposure to non-targeted tissues.20 21 In LSS, inflammation of neural and surrounding tissues contributes to mechanical allodynia and motor dysfunction. DEP’s anti-inflammatory properties may attenuate neuroinflammation, reduce neural sensitization, and alleviate pain hypersensitivity, thereby improving mechanical allodynia. Consequently, DEP exhibits stronger anti-inflammatory potency compared with free dexamethasone, with a reduced risk of steroid-related side effects due to its targeted delivery mechanism. On our histopathological examination, less epineurial inflammation or fibrosis was observed in the epidural DEP groups, which supports its anti-inflammatory effects.
Frequent epidural steroid injections are associated with various systemic side effects, including chemical meningitis, spinal cord embolism, impaired blood glucose control, increased fracture risk, adrenal suppression, and decreased immunity.22 23 While dexamethasone epidural injections have shown no significant increase in fasting blood glucose, they do elevate blood glucose levels significantly.24 This suggests that injecting an appropriate dose of glucocorticosteroid into the epidural space is generally safe, although with temporary effects on blood glucose. Steroid binds to glucocorticoid receptors (GRs) in neural and non-neural tissues, regulating gene expression and cellular signaling pathways involved in inflammation, apoptosis, and neuroplasticity. Activation of GRs by steroids may modulate neuronal excitability, synaptic transmission, and neuroplasticity in the spinal cord and peripheral nerves, contributing to the resolution of mechanical allodynia and restoration of motor function. In our study, DEP exhibited prolonged pain relief compared with dexamethasone. Despite the effectiveness of dexamethasone for epidural injections, long-term use poses potential risks. Therefore, DEP, an alternative to dexamethasone, may offer a safer and more effective long-term solution for epidural therapy with reduced frequency of administration.
Conclusions
Within the administered safe dose, DEP emerges as a viable option for epidural injection. In our study, we found no histological evidence of neurotoxicity or inflammation in the DRG, spinal cord, or adrenal gland following DEP injection in experimental rats. However, further investigation is needed to evaluate potential side effects associated with multiple doses of DEP.
Data availability statement
Data sharing not applicable as no datasets generated and/or analyzed for this study. No data are available.
Ethics statements
Patient consent for publication
Ethics approval
Institutional Animal Care and Use Committees of Seoul National University Bundang Hospital approved the study (IACUC No. BA-2305-367-003).
References
Footnotes
Contributors MHL: conducted experiments and contributed to manuscript writing. HYZ: contributed to manuscript preparation. EJC: contributed to manuscript preparation. FSN: validated the data. GYC: conducted investigation. PBL: supervised the study and acted as the guarantor.
Funding The authors have not declared a specific grant for this research from any funding agency in the public, commercial or not-for-profit sectors.
Competing interests None declared.
Provenance and peer review Not commissioned; externally peer reviewed.