Evaluation of an oral Beta-caryophyllene Indena Phytosome ® for the treatment of pain and inflammation in interstitial cystitis

Abstract

Background

Interstitial cystitis (IC), a chronic inflammatory condition of the urinary bladder characterized by pain and difficulties with urination, has limited treatment options. Beta-caryophyllene (BCP) is a sesquiterpene with anti-inflammatory and analgesic properties, but has poor water solubility and limited intestinal absorption which may limit oral bioavailability. Indena Phytosome^® is a proprietary food-grade lecithin-based delivery system that optimizes the bioavailability and pharmacokinetic profile of natural phytochemicals like BCP by producing a stable solid dispersion suitable for oral delivery.

Objective

To examine the therapeutic potential of an oral BCP Indena Phytosome® formulation (BCP Indena Phytosome^®) for the treatment of IC.

Methods

Female BALB/c mice were pre-treated with BCP Indena Phytosome^® or empty Indena Phytosome^® vehicle, then IC was induced using lipopolysaccharide. 24 h later, inflammation was evaluated using intravital microscopy and histology. Pain was evaluated using behavior scoring and von Frey asethesiometry.

Results

Pre-treatment with BCP Indena Phytosome^® reduced leukocyte adhesion, histology scores, and behavior scores in IC mice, indicating a reduction in inflammation and pain. Lipopolysaccharide administration did not alter leukocyte rolling, capillary perfusion, or von Frey scores.

Conclusions

Oral BCP Indena Phytosome^® effectively reduces pain and inflammation in experimental IC by decreasing leukocyte adhesion and extravasation in the bladder.

Keywords

inflammation interstitial cystitis intravital microscopy pain Indena Phytosome®terpenes

Introduction

Interstitial cystitis (IC), also known as bladder pain syndrome (BPS) or IC/BPS, is a chronic inflammatory disease of the bladder with unknown etiology.¹ IC/BPS is characterized by urothelial breakdown and bladder damage, as well as an increased release of inflammatory mediators and activation of mast cells, both of which increase bladder sensitization and painful symptoms.^2–9 In rare cases, this chronic inflammation causes fibrosis, which decreases bladder compliance, reduces urinary capacity, and increases urinary frequency.^6,10 Painful symptoms negatively impact mental and physical health, leading to significantly worse quality of life.^11–14 Prolonged stress increases inflammation and symptom severity, further exacerbating this process.¹⁵ IC/BPS has no cure, so treatments focus on controlling symptoms while minimizing side effects. First-line treatment is non-invasive lifestyle changes, but many patients require pharmacological treatments that disrupt the cycle of damage.^6,16 When pharmacological treatments fail, more invasive treatments such as cystectomy are employed.¹⁶ IC/BPS is more common in women, with an estimated prevalence of 2.70–6.53% in women compared to 1.9–4.2% in men.^17,18

Cannabinoid receptor agonists are an emerging treatment strategy to decrease pain and inflammation through modulation of the endocannabinoid system, a homeostatic body system mediated by the G protein-coupled receptors cannabinoid receptor 1 (CB1) and cannabinoid receptor 2 (CB2).^16,19–21 CB1 receptors are highly expressed in the CNS, with lower levels in the periphery.^22,23 In contrast, CB2 receptors are largely found in the immune system and periphery, including on immune cells and various organs.^22,24,25 CB2 receptors are upregulated in response to inflammation and mediate the immune response without the undesirable cardiovascular, immune, and CNS-related side effects seen with CB1 activation.^{19,22,26–28} CB2-specific agonists showed anti-inflammatory activity in a model of IC by reducing endothelial infiltration of leukocytes and mRNA expression of inflammatory cytokines.²⁹ In addition, CB2 activation has analgesic effects, particularly in models of inflammatory and neuropathic pain.^27,30,31 These anti-inflammatory and analgesic effects, combined with CB2 localization on both bladder and immune cells,^24,25 mean that CB2 receptors are an excellent target to mitigate the inflammatory dysfunction that drives IC/BPS.

Beta-caryophyllene (BCP) is a generally recognized as safe (GRAS) non-psychoactive sesquiterpene found in various essential oils and plants, including Cannabis sativa.^32,33 However, BCP is also an emerging anti-inflammatory and analgesic agent that functions primarily through activation of CB2 receptors, with contributions from peroxisome proliferator-activated receptor alpha (PPARα), peroxisome proliferator-activate receptor gamma (PPARγ), and transient receptor potential vanilloid type-1 (TRPV1).^30,33–36 BCP treatment via various routes can reduce bladder inflammation and pain, without the development of tolerance to its analgesic effects.^30,32 BCP has also shown short-lived local anesthetic activity both in vitro and in vivo.³⁷

BCP's use as a therapeutic agent, especially in an oral formulation, is challenging due to its hydrophobic nature, strong aroma, and flavor. However, oral drug delivery is more convenient and less invasive for patients. Encapsulation of essential oil constituents as solids can increase stability and improve bioavailability.³⁸ We evaluated the proprietary Indena Phytosome^® formulation of BCP (BCP Indena Phytosome^®), a solid dispersion of BCP in a food-grade matrix that uses the natural surfactant lecithin to facilitate the absorption of lipophilic compounds.^39–43 The phospholipids in the Indena Phytosome^® formulation interact with BCP to prevent self-aggregation and generate a stable, solid dispersion that can be delivered via oral capsules. This improves the physical and chemical stability and dissolution profile for BCP and optimizes the absorption rate and pharmacokinetic profile for the phytonutrient ingredients.⁴¹ The BCP Indena Phytosome^® has no pharmacological adjuvants, providing an excellent safety profile.⁴²

Building on previous work from our group,³⁰ the objective of this study was to determine whether the proprietary BCP Indena Phytosome^® formulation could reduce pain and inflammation in a murine model of IC. To assess inflammation, we compared local inflammatory changes in the mouse bladder after LPS administration with and without oral BCP treatment using intravital microscopy (IVM) as well as H&E staining of the bladder. We then assessed the analgesic effects of BCP on IC in vivo using von Frey aesthesiometry and a behavior scoring scale.

Materials and methods

Animals

All experimental procedures were approved by the University Committee on Laboratory Animals (protocol 21-032), in accordance with the guidelines developed by the Canadian Council on Animal Care. Female BALB/c mice (8–12 weeks) were purchased from Charles River Laboratories International (Wilmington, Mississippi, USA) and housed in the Carleton Animal Care Facility (CACF) in ventilated, climate-controlled rack cages on a 12 h light/dark cycle with ad libitum access to water and standard mouse chow. All mice were acclimatized to the CACF for a minimum of one week before experimental procedures.

Interstitial cystitis induction and experimental groups

Fifteen minutes before IC induction, all the mice were treated via oral gavage with 100 mg/kg body weight (5 µl/g) of the oral BCP Indena Phytosome^® (Indena, Milan, Italy) in corn oil (density: 0.92 g/cm³; Sigma-Aldrich, ON, Canada), or the same volume of corn oil as vehicle control (VEH) or empty Indena Phytosome^® (Indena, Milan, Italy) in corn oil as another control (PHY). Phytosome™ is a trademark of Indena, S.p.A., Italy. Mice were then anesthetized with 5% isoflurane and maintained at 1.8–2.2% isoflurane. The mouse was placed on a heating pad in a supine position, with a piece of tape securing the position of the nose cone connected to the isoflurane. The mouse was monitored every five minutes via pedal reflex to ensure surgical depth of anesthesia.

Once the mouse was under anesthesia, the suprapubic area of the mouse was gently pressed to empty any remaining urine from the bladder. Then, the urethral opening was sanitized using an alcohol swab and 4 cm of gas sterilized polyethylene P10 tubing (inner diameter: 0.28 mm, BD Intramedic, Sparks, MD, USA) lubricated with vaseline was inserted into the bladder via the urethra. The tubing was connected to a 1 ml syringe through a 30-gauge needle (0.3 mm×13 mm, BD PrecisionGlide, Franklin Lakes, NJ, USA). The tubing, needle and syringe were prefilled with either normal saline (as SHAM) or Escherichia coli (E. coli) LPS (150 µg/ml, 0.375 mg/kg; serotype O26:B6, L8274; Sigma-Aldrich, ON, Canada) and 50 µl of the solution was given over a period of ten seconds to minimize vesicoureteral reflux.³⁰ Then the tubing was carefully removed, and the urethral opening was held shut with an aneurysm clip to maintain the liquid in the bladder for 30 min. Then the aneurysm clip was removed, and the mouse was placed back to a clean cage to recover for approximately 24 h before endpoint measurements were taken. This resulted in six total experimental groups: SHAM VEH, SHAM PHY, SHAM PHY + BCP, LPS VEH, LPS PHY, LPS PHY + BCP (Table 1).

Table 1.

Experimental groups.

Group	Oral treatment	Intravesical instillation
SHAM VEH	Corn oil	Saline
SHAM PHY	Empty Indena phytosome^®in corn oil	Saline
SHAM PHY + BCP	BCP Indena phytosome^® in corn oil	Saline
LPS VEH	Corn oil	LPS
LPS PHY	Empty Indena phytosome^® in corn oil	LPS
LPS PHY + BCP	BCP Indena phytosome^® in corn oil	LPS

Intravital microscopy

Anesthesia and surgery

23.5 h after IC induction, mice were anesthetized with an i.p. injection of pentobarbital (90 mg/kg, 27.3 mg/ml; Ceva Sante Animale, Montreal, QC, Canada). Mice were placed on a reflection heating pad to maintain their body temperature at 37°C for the duration of imaging. Depth of anesthesia was confirmed by pedal reflex and maintained by repeated administration of pentobarbital (i.p. 0.1–0.2 mL of 5.47 mg/ml). Then a mix of rhodamine 6G (1.5 ml/kg, 0.75 mg/kg body weight; Sigma-Aldrich, ON, Canada), used to visualize leukocytes, and fluorescein isothiocyanate (FITC)-albumin (1 ml/kg, 50 mg/kg; Sigma-Aldrich, ON, Canada), used to visualize capillaries, was administered via tail vein injection.

The mouse was placed on its back and a midline incision of the lower abdomen was made to expose the abdominal cavity. The gut and surrounding tissues were moved aside using wet cotton tip applicators to fully expose and exteriorize the bladder from the abdominal cavity. Care was taken to minimize damage to surrounding tissues, including avoiding cutting excess fat whenever possible. Any remaining urine was emptied by gently squeezing the bladder with wet cotton tip applicators. The bladder was kept moist by frequent administration of saline.

After exteriorization, the bladder was slowly filled with 100 µl of saline using sterilized tubing connected to 1 ml syringe via 30 g needle (as described above for IC induction). Immediately after the tubing was removed from the urethra, an aneurysm clip was used to hold the urethral opening shut and keep the bladder full for the duration of imaging. The bladder was moistened with saline, and an 18 mm round glass coverslip was placed on top to facilitate imaging. Then, the mouse and heating pad were transferred over to the microscope stage for imaging.

Microscopy

Imaging of bladder microcirculation took place 24 h after IC induction using an epifluorescent microscope (Leica, DM LM, Wetzlar, Germany) with the 20x objective lens (Leica, Germany) and a 10x eyepiece (HC Plan, Leica, Germany). Green light, passing through a 530–550 nm bandpass excitation filter (which excites rhodamine at an emission wavelength of 515 nm) was used to visualize leukocyte trafficking in venules. Blue light passing through a 460–490 nm bandpass excitation filter (which excites FITC at an emission wavelength of 520 nm) was used to allow visualization of functional capillary density (FCD) in the microcirculation. A minimum of six visual fields per light color were captured for a period of 30 s each using Volocity software (Perkin Elmer, Waltham, MA, USA).

Video analysis

IVM videos were captured using a digital EM-CCD camera C9100-02 with AC-adapter A3472-07 (Hamamatsu, Herrsching, Germany) and analyzed using ImageJ software (NIH, USA). All videos were analyzed in a blinded fashion.

Leukocyte adhesion was determined by outlining a section of a submucosal venule, which was then calculated as a cylindrical surface area. The number of leukocytes within that area that did not move over the 30 s capture period (defined as “adherent leukocytes”) were counted and divided by the total area to give a measurement of number of adherent leukocytes per mm².

Leukocyte rolling was determined by quantifying the number of leukocytes that passed a set point (or an assumed line) over the 30 s capture period. A random submucosal venule was chosen, and a line was drawn across its lumen using ImageJ software. The number of leukocytes passing that line was counted and converted to a value of number of rolling leukocytes per minute.

FCD was determined by measuring capillary perfusion over a set area. A rectangular section of the video area was marked, and the area measured. Within that area, the distance each capillary that perfused over the 30 s capture period was marked using the ImageJ software and the area measured. The distance each capillary perfused was summed and divided by the total area observed to give a measurement of FCD in cm/cm².

Pain assessment

Acclimatization

Mice were acclimatized to the von Frey enclosure, consisting of an elevated mesh floor and a clear plexiglass separator (IITC Life Sciences, Woodland Hills, CA, USA) for 15 min per day on the two days leading up to the experimental procedures. The enclosure was in a dim, quiet room with only the observer present. On the second day of acclimatization, mice were poked in the lower abdomen three times using the von Frey filament. Preceding the pre-treatment and IC induction on the third day, mice were placed in the von Frey enclosure to acclimatize for a minimum of one hour before the baseline pain assessments were performed. Mice were also acclimatized for minimum one hour before the endpoint pain assessment.

Behavior scoring

Preceding IC induction and the experimental endpoint, mouse behavior was observed based on its eye opening, posture, and motoric activity before the acclimatization. A blinded examiner scored each individual parameter separately from 0–10, with 0 indicating no evidence of pain and 10 being maximum evidence of pain (Table 2). The sum of the three parameter scores was defined as the behavior score and the overall change in behavior score between the beginning and endpoint of the experiment was used as an indicator for non-evoked pain.

Table 2.

Behavior scoring scale. Individual parameters for each mouse were scored before and after cystitis induction according to the description, then totaled to give a score out of 30. Levels of eye opening and posture that fell between the extremes were given intermediate, whole number scores.

Parameter	Description
Eye opening	0: eyes fully open 5: eyes halfway open 10: eyes fully closed
Posture	0: normal posture 10: fully rounded back or limp posture
Motoric activity	Level of activity within 20 s; every 2 s spent not moving adds one point to the score (2 s of stillness/20 s = score of 1/10)

von Frey aesthesiometry

Following the acclimatization and behavior assessment, evoked pain was measured using a 2390 series IITC Life Science Electronic von Frey aesthesiometer (Woodland Hills, California, USA). Using the rigid tip, the von Frey aesthesiometer was applied to the lower abdomen of the mouse with increasing force until the mouse withdrew. The maximum force tolerated before withdrawal (in grams) was recorded. The sum of five individual measurements per mouse (with a minimum of 30 s between each measurement) was recorded and the overall change between the beginning and endpoint of the experiment was used to indicate evoked pain.

Histology and histopathology scoring

At the end of experiments, mice were sacrificed by a pentobarbital overdose followed by cervical dislocation. The bladder was removed and immersed in 10% neutral buffered formalin. Bladders were then processed for histopathology scoring, which included dehydration, embedding, slicing (5 µm cross-sections), and staining with hematoxylin and eosin (H&E).

The H&E slides were viewed on an Optika B-290TB brightfield microscope (Optika Microscopes, Ponterica, Italy) with the 40x objective. Samples were scored based on a scoring system adapted from Hopkins et al. 1998,⁴⁴ with modifications based on suggestions from Dr Cheng Wang (Departments of Pathology and Urology, Dalhousie University, Halifax, Nova Scotia, Canada), as seen in Table 3.

Table 3.

Histology scoring scale.

Score	Criteria
0	No signs of inflammation; inflammatory cells primarily contained within vessels.
1	Focal infiltration of inflammatory cells into the subepithelium.
2	Criteria of score 1 plus multifocal infiltration of inflammatory cells in the subepithelium; may have signs of mild edema.
3	Criteria of score 2 plus diffuse infiltration of inflammatory cells in the subepithelium and edema; may have signs of neutrophil necrosis.
4	Criteria of score 3 plus infiltration of inflammatory cells into the mucosa.
5	Criteria of score 4 plus infiltration of inflammatory cells into the muscle layer.
6	Criteria of score 6 plus loss of surface epithelium.

Statistical analysis

All statistical analysis was completed using GraphPad Prism 10 (GraphPad Software, La Jolla, CA, USA). Data was first analyzed for normality using the Kolmogorov-Smirnov Test. Outliers were removed using the ROUT method. Data was analyzed for significance using a one-way ANOVA with Dunnett's test for post-hoc analysis. Comparisons between two groups were analyzed using a one- or two-tailed t-test. A p-value of less than 0.05 was considered statistically significant and data was expressed as mean plus or minus standard deviation.

Results

Oral BCP Indena Phytosome^® decreases lipopolysaccharide-induced bladder inflammation

Intravital microscopy

The oral BCP Indena Phytosome^® was effective at reducing inflammation within the bladder microvasculature as evaluated by IVM (Figure 1). LPS administration significantly increased leukocyte adhesion (p < 0.05) in the bladder microvasculature of mice compared to control when observed 24 h after experimental IC induction. When mice were pre-treated with 100 mg/kg of oral BCP Indena Phytosome^®, leukocyte adhesion was significantly decreased (p < 0.05) compared to untreated LPS controls. IC mice treated with the empty Indena Phytosome^® vehicle control showed decreased leukocyte adhesion in the bladder microvasculature compared to untreated mice, but this difference was not statistically significant. No significant differences were observed between sham groups.

Figure 1.

100 mg/kg of Indena Phytosome^® BCP reduces leukocyte adhesion in submucosal bladder venules of female BALB/c mice 24 h after IC induction. Leukocyte adhesion was assessed using intravital microscopy in the following groups: sham IC with vehicle control (SHAM VEH), sham IC with empty Indena Phytosome^® (SHAM PHY), sham IC with BCP Indena Phytosome^® (SHAM PHY + BCP), IC with vehicle control (LPS VEH), IC with empty Indena Phytosome^® (LPS PHY), and IC treated with BCP Indena Phytosome^® (LPS PHY + BCP). Data is in cells/mm² and is represented as mean ± SD, n = 5/group. Statistical analysis by one-way ANOVA with multiple comparisons, *p < 0.05.

No significant differences were observed between groups regarding leukocyte rolling behavior and FCD. There were no differences between sham groups or with untreated LPS compared to both control and treatment groups with either leukocyte rolling (Figure 2) or FCD (Figure 3). Representative IVM images can be found in Figure 4.

Figure 2.

LPS administration (0.375 mg/kg) does not alter leukocyte rolling in submucosal bladder venules of female BALB/c mice after 24 h. Leukocyte rolling was assessed using intravital microscopy in the following groups: sham IC with vehicle control (SHAM VEH), sham IC with empty Indena Phytosome^® (SHAM PHY), sham IC with BCP Indena Phytosome^® (SHAM PHY + BCP), IC with vehicle control (LPS VEH), IC with empty Indena Phytosome® (LPS PHY), and IC treated with BCP Indena Phytosome^® (LPS PHY + BCP). Data is in cells/min and is represented as mean ± SD, n = 5/group.

Figure 3.

LPS administration (0.375 mg/kg) does not alter functional capillary density in the bladder microvasculature of female BALB/c mice after 24 h. Functional capillary density was assessed using intravital microscopy in the following groups: sham IC with vehicle control (SHAM VEH), sham IC with empty Indena Phytosome^® (SHAM PHY), sham IC with BCP Indena Phytosome^® (SHAM PHY + BCP), IC with vehicle control (LPS VEH), IC with empty Indena Phytosome^® (LPS PHY), and IC treated with BCP Indena Phytosome^® (LPS PHY + BCP). Data is in cm/cm² and is represented as mean ± SD, n = 5/group.

Figure 4.

Still-frame intravital microscopy images of leukocyte adhesion (A and B) and functional capillary perfusion (C and D) within the bladder microcirculation of female BALB/c mice. (A) a control animal with few adherent leukocytes. (B) an untreated LPS animal with many adherent leukocytes. (C) a control animal with dense microvasculature. (D) an untreated LPS animal with reduced number of perfused capillaries. Arrows indicate adherent leukocytes. Images taken at a magnification of 200x (20x objective and 10x eyepiece). Scale bars indicate 150 µm.

Bladder histopathology

Oral BCP Indena Phytosome^® was effective at reducing histological signs of inflammation within the mouse bladder wall (Figure 5). LPS administration significantly increased histology scores (p < 0.01) in mouse bladders 24 h after experimental IC induction. When mice were pre-treated with 100 mg/kg of the oral BCP Indena Phytosome^®, histology scores were significantly decreased (p < 0.05) compared to untreated LPS controls. Bladders from the IC mice treated with the empty Indena Phytosome^® had histology scores that fell between the scores for untreated and the BCP Indena Phytosome^®-treated groups, but the difference was not statistically significant from either group. No significant differences were observed between sham groups. Representative histology images showing normal and inflamed bladders are shown in Figure 6.

Figure 5.

BCP Indena Phytosome^® normalizes histological signs of inflammation in female BALB/c mouse bladders 24 h after IC induction. Histological changes were assessed in the following groups: sham IC with vehicle control (SHAM VEH; n = 5), sham IC with empty Indena Phytosome^® (SHAM PHY; n = 5), sham IC with BCP Indena Phytosome^® (SHAM PHY + BCP; n = 7), IC with vehicle control (LPS VEH; n = 5), IC with empty Indena Phytosome^® (LPS PHY; n = 5), and IC treated with BCP Indena Phytosome^® (LPS PHY + BCP; n = 5). Data is represented as mean ± SD. Statistical analysis by one-way ANOVA with multiple comparisons, **p < 0.01, and one-tailed t-test, #p < 0.05.

Figure 6.

Representative histology images of female BALB/c mouse bladders. Images represent (A) a control animal (representative score of 0) and (B) an untreated LPS animal (arrow indicates edema with diffuse immune cell infiltration in the subepithelium; representative score of 3) at a magnification of 100x. Scale bars indicate 100 µm.

Oral BCP Indena Phytosome^® decreases lipopolysaccharide-induced pain

Behavior scoring

The oral BCP Indena Phytosome^® treatment was effective at reducing behavioral signs of pain (Figure 7). LPS administration significantly increased behavior scores (p < 0.01) compared to control 24 h after experimental IC induction. When mice were pre-treated with 100 mg/kg of the oral BCP Indena Phytosome^®, behavior scores were significantly decreased (p < 0.05) compared to untreated LPS controls. While the IC mice treated with the empty Indena Phytosome^® appeared to have elevated behavior scores compared to control, this difference was not statistically significant. No significant differences were observed between sham groups.

Figure 7.

BCP Indena Phytosome^® reduces behavioral signs of pain in female BALB/c mice 24 h after ic induction. Behavioral signs of pain were assessed in the following groups: sham IC with vehicle control (SHAM VEH; n = 5), sham IC with empty Indena Phytosome^® (SHAM PHY; n = 4), sham IC with BCP Indena Phytosome^® (SHAM PHY + BCP; n = 5), IC with vehicle control (LPS VEH; n = 5), IC with empty Indena Phytosome^® (LPS PHY; n = 5), and IC treated with BCP Indena Phytosome^® (LPS PHY + BCP; n = 8). Each data point represents the change in behavior score 24 h after IC induction compared to the individual mouse's initial behavior score. Data represented as mean ± SD. Statistical analysis by one-way ANOVA with multiple comparisons, *p < 0.05, **p < 0.01.

von Frey aesthesiometry

Overall, there were no significant differences in evoked pain response when evaluated by one-way ANOVA (Figure 8). LPS did induce a significant reduction in force tolerated on the lower abdomen (*p < 0.05) when compared to control using a two-tailed t-test. However, treatment with BCP Indena Phytosome^® did not significantly alter this change. The the BCP Indena Phytosome^®-treated group seemed to have decreased evoked pain tolerance after IC induction compared to control, while the LPS treated with empty Indena Phytosome^® appeared to have a higher evoked pain tolerance compared to the other LPS groups, but these differences were not statistically significant. There were no differences between sham groups.

Figure 8.

LPS administration (0.375 mg/kg) does not alter evoked pain tolerance of female BALB/c mice 24 h after IC induction. Behavioral signs of pain were assessed in the following groups: sham IC with vehicle control (SHAM VEH; n = 5), sham IC with empty Indena Phytosome^® (SHAM PHY; n = 5), sham IC with BCP Indena Phytosome^® (SHAM PHY + BCP; n = 5), IC with vehicle control (LPS VEH; n = 5), IC with empty Indena Phytosome^® (LPS PHY; n = 5), and IC treated with BCP Indena Phytosome^® (LPS PHY + BCP; n = 8). Each data point represents the change in force tolerated before withdrawal 24 h after IC induction compared to the individual mouse's initial tolerance. Data is in grams and is represented as mean ± SD.

Discussion

Pre-treatment with oral BCP Indena Phytosome^® reduced signs of inflammation and pain in the present model. Decreased leukocyte adhesion was observed in the microvasculature of the bladder wall in treated IC mice compared to untreated IC mice, indicating that BCP Indena Phytosome^® decreases immune cell activation. There was no change in leukocyte rolling or capillary perfusion. Reductions in bladder histology scores after pre-treatment with the oral BCP Indena Phytosome^® indicated a reduction in leukocyte infiltration and edema.

Furthermore, pre-treatment with BCP Indena Phytosome^® decreased behavior scores in IC mice, indicating that this treatment may decrease inflammatory pain. Overall pain levels were not high on our scale, as untreated IC mice demonstrated an average score increase of 4 out of a total possible increase of 30, but pain levels were significantly increased compared to both sham and treated mice. Additionally, these pain levels fit with the modest but significant signs of inflammation demonstrated in the IVM and histology studies. Though BCP Indena Phytosome^® pre-treatment did not return evoked pain levels to baseline, the difference in evoked pain tolerance was significant when comparing only the untreated LPS group to the sham group. Taken together, these findings indicate that intravesical administration of 0.375 mg/kg of LPS induces mild inflammation and pain in vivo, and that this inflammation and pain are reduced with oral pre-treatment of 100 mg/kg BCP Indena Phytosome^®.

The reduction in leukocyte adhesion and histology scores by the BCP Indena Phytosome^® supports the growing body of evidence that BCP is an effective anti-inflammatory agent. In both infectious and non-infectious models of cystitis, BCP reduces leukocyte adhesion and increases capillary perfusion, indicating decreased inflammation.^30,45 Since immune cells express CB2 receptors,²⁵ BCP could be acting directly on leukocytes to decrease their adhesion and infiltration. One study found that CB2-activated leukocytes that were injected into mice showed decreased adhesion and infiltration across the blood–brain barrier.⁴⁶ This may be explained by changes in the expression of cellular adhesion molecules, as BCP treatment decreases the levels of E-selectin, P-selectin, ICAM-1, and VCAM-1 in various inflammatory models.^35,47 Other studies have used CB2 activation to decrease immune cell infiltration in the bladder, with Tambaro et al. reporting a significant decrease in leukocytes when IC mice were treated with the CB2 agonist JWH015, and a reversal of this effect with the CB2 receptor antagonist AM630.²⁹

BCP could also be acting directly on the bladder epithelial cells, which also express CB2 receptors.²⁴ Intravesical CB2 agonist administration can improve bladder contraction, decrease bladder pressure, and increase time between voids.^24,48 However, in contrast to the present study, these functionality studies did not examine BCP's anti-inflammatory effects directly, so further work into BCP's effects on the urothelium is required.

Receptors other than CB2 could also be contributing to BCP's anti-inflammatory effects. A study examining the anti-inflammatory effects of BCP (100 mg/kg i.p.) in a model of UTI indicated that BCP reduced signs of inflammation in the bladder, but that these changes were not reversed with administration of AM630, indicating that mechanisms other than CB2 activation must be contributing to BCP's anti-inflammatory effects in bacterial cystitis.⁴⁵ Although PPARγ involvement has not been confirmed in cystitis, BCP has shown anti-inflammatory effects in models of vascular inflammation and neuroinflammation through activation of PPAR in conjunction with CB2.^35,49

Beyond BCP itself, the components of Indena Phytosome^® may be contributing to the anti-inflammatory effects observed. While the specific mechanism(s) by which Indena Phytosome^® improves the absorption of plant phenolics and terpenoids is unclear, several processes have been proposed, including improved dispersion of these phytochemicals in the intestinal fluids and a process whereby lecithin “chaperones” the phytochemicals into the enterocytes.^41,43 In the present work, the IC animals that received Indena Phytosome^® alone had leukocyte adhesion levels and histology scores that fell between, but did not differ significantly from, both corn oil-treated and BCP Indena Phytosome^® treated IC groups. Phosphatidylcholines, a primary component of the Indena Phytosome^®, have demonstrated anti-inflammatory effects. Orally administered phosphatidylcholines were anti-inflammatory in a rat model of knee arthritis, reducing leukocyte adhesion and infiltration and decreasing ICAM-1 expression.⁵⁰ Beyond their effects on immune cells, phosphatidylcholines have also restored intestinal barrier function after liver injury in rats.⁵¹ While we did not examine barrier function directly, bladder histology scores, which take into account epithelial damage, also appeared lower when IC mice were given Indena Phytosome^® alone.

The lack of LPS-induced change to both leukocyte rolling and capillary perfusion is in line with other studies that used IVM to evaluate LPS-induced changes in the bladder microcirculation. Studies using a 2 h endpoint and 0.375 mg/kg of LPS found mixed results for capillary perfusion.^30,52 However, work by Kowalewska et al. indicates no change in leukocyte rolling 4 h after administration of 1 mg/kg of LPS.⁵³ Overall, these LPS -induced changes are likely time and dose dependent, but further work is needed to confirm.

The decrease in behavior score with oral BCP Indena Phytosome^® treatment fits with previous work on BCP's effects on pain. In a model of formalin-induced inflammatory pain, 5 mg/kg of oral BCP reduced behavioral signs of pain in the hindpaw.³² In an acute cystitis model, Berger et al. found that 100 mg/kg of oral BCP significantly reduced behavioral signs of pain compared to control 2 h after LPS administration, although unlike the present work, their BCP-treated group still had significantly increased pain levels compared to non-IC controls.³⁰ While Berger et al. used a shorter timepoint and a different vehicle than the present work, this indicates that the BCP Indena Phytosome^® may be a more effective analgesic than BCP alone.

There are several potential mechanisms for these pain-relieving effects. BCP has been shown to attenuate neuropathic pain in a CB2-dependent manner, as administration of AM630 reversed BCP's effects on mechanical allodynia.³¹ BCP also mediates local anesthetic effects and acts on sodium channels, which is the mechanism by which the local anesthetic lidocaine produces pain-relieving effects^34,37,54 It is also possible that TRPV1 and/or PPARα could be contributing to BCP's analgesic effects, as black pepper seed extract containing 30% BCP effectively reduced pain through CB2, TRPV1, and PPARα activation, with molecular docking studies predicting strong binding affinity with the agonist-binding sites of all three of these receptors.³⁴

It was suggested that Indena Phytosome^® components could produce additional analgesic effects. One study found that an empty liposome vehicle, made of phosphatidylcholines from an egg, showed no analgesic effect in an aversive response of rat upper lip pinching.⁵⁵ Conversely, another study found that arthritic pain tolerance was significantly increased with treatment with oral phosphatidylcholines.⁵⁰ Taken in conjunction with the results of the present study, it seems that the analgesic effect of Indena Phytosome^® vehicle alone may be explained by the small reduction in inflammation observed with this treatment condition, but further research is required to confirm the exact mechanism.

Despite seeing a clear effect on behavior scoring, the results for evoked pain tolerance using von Frey aesthesiometry did not follow the same pattern. We saw no significant differences in the force tolerated between groups with a one-way ANOVA. We saw a modest reduction in force tolerated upon LPS administration, which indicates an increase in pain, but we did not see any significant differences between any of the other groups, and there was a large amount of within-group variation. Taken in conjunction with findings from Berger et al., who used the same LPS dose as the present study at a shorter timepoint and also observed no change in force tolerated, this suggests that it is possible that there was not enough inflammation to cause a large enough difference in pain tolerance to treat.³⁰ Work by Dickson et al. supports this, as their von Frey results for a murine model of UTI were variable within groups, especially 6 h after infection.⁴⁵ However, earlier studies found that abdominal von Frey was a reliable measure of pain, as both Berger et al. and Hagn et al. used this same method and observed consistent results within groups.^30,52

In conclusion, we found that the proprietary oral BCP Indena Phytosome^® formulation is effective at reducing both leukocyte adhesion and extravasation in the microvasculature of the bladder wall and attenuating behavioral signs of pain in experimental IC in vivo. The observed anti-inflammatory and analgesic effects of oral BCP Indena Phytosome^® represent a potential avenue for the treatment of patients with IC.

Footnotes

Acknowledgements

The authors acknowledge the support of Indena, including the use of their proprietary Indena Phytosome® formulation, as well as the support of Matthew Allain, Bithika Ray, Tanya Myers, and Cheng Wang.

Ethical considerations

All experimental procedures were approved by the Dalhousie University Committee on Laboratory Animals (protocol 21-032), in accordance with the guidelines developed by the Canadian Council on Animal Care.

Consent to participate

N/A.

Consent for publication

N/A.

Author contributions

Conceptualization, A.C., J.Z., M.K. and C.L.; methodology, J.Z., C.L.; investigation, H.W., K.D., C.S., D.N.; formal analysis, H.W.; writing—original draft, H.W.; writing—review and editing, H.W., K.D., M.K., and C.L.; funding acquisition, J.Z., M.K. and C.L.; supervision, J.Z., M.K. and C.L.

Funding

This research was funded by MITACS Canada.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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Evaluation of an oral Beta-caryophyllene Indena Phytosome ® for the treatment of pain and inflammation in interstitial cystitis

Abstract

Background

Objective

Methods

Results

Conclusions

Keywords

Introduction

Materials and methods

Animals

Interstitial cystitis induction and experimental groups

Intravital microscopy

Anesthesia and surgery

Microscopy

Video analysis

Pain assessment

Acclimatization

Behavior scoring

von Frey aesthesiometry

Histology and histopathology scoring

Statistical analysis

Results

Oral BCP Indena Phytosome® decreases lipopolysaccharide-induced bladder inflammation

Intravital microscopy

Bladder histopathology

Oral BCP Indena Phytosome® decreases lipopolysaccharide-induced pain

Behavior scoring

von Frey aesthesiometry

Discussion

Footnotes

Acknowledgements

Ethical considerations

Consent to participate

Consent for publication

Author contributions

Funding

Declaration of conflicting interests

References

Oral BCP Indena Phytosome^® decreases lipopolysaccharide-induced bladder inflammation

Oral BCP Indena Phytosome^® decreases lipopolysaccharide-induced pain