Co-Administration of nalbuphine to improve morphine tolerance in mice with bone cancer pain

Abstract

Background

Kappa-opioid receptor (KOR) agonists are known for having opposite and/or different effects compared with Mu-opioid receptor (MOR) agonists. This study is aimed at clarifying the analgesic effect and tolerance of nalbuphine combined with morphine, and quantifying the mRNA and protein expression of spinal MOR and KOR in a mouse bone cancer pain (BCP) model treated with nalbuphine and morphine.

Method

BCP model was prepared in C3H/HeNCrlVr Mice by implanting the sarcoma cells into the intramedullary space of the femur. The paw withdrawal thermal latency (PWL) measured by thermal radiometer was used to assess thermal hyperalgesia. PWL testing was performed after implantation and drug administration according to the protocol. Hematoxylin-eosin staining in the spinal cord and x-ray in the femoral intramedullary canal was detected. Real-time PCR and western blot analysis played a role in detecting spinal MOR and KOR expression changes.

Results

In tumor-implanted mice, the spinal MOR and KOR protein and mRNA expression was down-regulated when compared to that in sham-implanted mice (p < 0.05). Morphine therapy can lead to a decrease in spinal μ receptor expression. Similarly, the nalbuphine therapy can lead to a decrease in the expression of κ receptor protein and mRNA at the spinal cord level (p < 0.05). Morphine, nalbuphine, or nalbuphine co-administration with morphine all can extend the paw withdrawal thermal latency (PWL) to radiant thermal stimulation in tumor-implanted mice (p < 0.05). Compared with the morphine treatment group, nalbuphine co-administration with morphine delayed the reduction of PWL value again (p < 0.05).

Discussion

BCP itself may induce down-regulation of the spinal MOR and KOR expression. A low dose of nalbuphine co-administration with morphine led to the delayed emergence of morphine tolerance. The part of the mechanism may be due to the regulation of spinal opioid receptors expression.

Introduction

The prevalence rate of pain related to cancer has been reported ranging from 50% to 75% in recent years, and one of the most common and suffering symptoms is pain from the bone metastasis. ¹ The survival time of patients diagnosed with malignant diseases improved by progressive diagnostic and therapeutic strategies, which increased the prevalence rates of both bone metastasis and the accompanying bone cancer pain (BCP). ² BCP usually happened to patients diagnosed with primary bone tumors or secondary metastatic bone tumors from breast, prostate, and lung cancer. ³ Opioids have been widely applied to the management of cancer pain, but its increasing dosage and frequency attribute to opioid tolerance continues to be an urgent problem. Tolerance refers to the declining of drug efficacy because of the long-term or frequent application, resulting in the reduction of drug effectiveness and improvements of drug dosage to preserve the ideal analgesic effects. ⁴ The high drug dosage may increase the incidence of side effects, which involved in respiratory depression, pruritus, constipation, and drug addiction. ⁵ According to the previous study, opioid receptors are classified into µ (Mu-opioid receptor, MOR), δ (Delta-opioid receptor, DOR), κ (Kappa-opioid receptor, KOR), and the non-classical nociception receptor (NOR). ⁶ Though all subtypes of opioid receptors had the effect on analgesia modulation to some extent, the MOR is considered as the major receptor to relief pain.^7,8 KOR agonists usually play opposite and/or different roles compared with MOR agonists. ⁹ Historically, for KOR agonists, there are potential clinical pharmacologists to perform acceptable analgesia, and there are also less morphine-like physical dependence, little respiratory depression and gastrointestinal transition. However, the frequent appearance of dysphoria has limited the centrally-action of KOR agonists.^10,11 The above-mentioned studies had implied that although there were different mechanisms for MOR and KOR agonists to perform the analgesic effects, the co-administration of MOR and KOR may attenuate some mutual pharmacological side effects. Nalbuphine, a synthetic agonist-antagonist opioid, acts as a KOR agonist and a partial MOR antagonist. Nalbuphine plays its analgesic effects of by mediating the agonist activity of the KOR. ¹² Compared to morphine, nalbuphine provides analgesic effects, but has fewer side effects of nausea, pruritus, and respiratory depression, owing to the complex agonist-antagonist properties. ¹³ Nalbuphine is one of the FDA-indicated opioids for moderate to severe pain when other alternative treatments have been inefficient. Although nalbuphine has proven to be potently against acute and chronic pain, its effect on BCP has not been explored.^14,15 Besides, it is widely known to apply morphine with opioid receptor agonist-antagonists to relief pain in pain management, but it remains unknown about the mechanism.^16,17 Nalbuphine exerts its reversal effects of pruritus induced by morphine without decreasing the analgesic effects, implying an action through KOR agonism.

Current study

This study aimed at exploring the analgesic effect and tolerance of nalbuphine in co-application of morphine in a mouse BCP model, by detecting the mRNA and protein expression of spinal KOR and MOR which may be induced partly by BCP itself, in order to provide preliminary data about the combination of nalbuphine and morphine therapy, and opioid receptor-related mechanisms of BCP.

Methods

The present experiments were approved by the Affiliated Hospital of Jiangnan University Animal Care Committee and the ethical guidelines of the National Institutes of Health. There are diverse efforts conducted to minimize the suffering and the number of animals in our study. Adult male C3H/HeNCrlVr mice weighing 20–25 g (Weitong Lihua Laboratory Animal Technology Co., Ltd, Beijing, China) were selected. The mice were raised in a temperature-consistent (21 ± 1°C) room, equipped with a 12-h light/dark cycle artificially, and fed with basic water and fodder by free access. Experiments were carried out according to the flow chart (Figure 1).

Figure 1.

Flow diagram of the experiment design.

Drugs and reagents

Morphine Hydrochloride was obtained from Northeast Pharmaceutical Group (China). Nalbuphine Hydrochloride Injection came from Yichang Humanwell Pharmaceutical Co., Ltd (China). MOR (OPRM1) Antibody (Cat. #: DF5045) and KOR (OPRK1) Antibody (Cat. #: DF5044) were purchased from Affinity Biosciences (USA). KOR and MOR mRNA Primer and SYBR Green Mix kit were obtained from Vazyme (China).

Bone cancer model

Murine sarcoma cells (NCTC 2472; ATCC, Rockville, MD, USA) were maintained in NCTC 135 media (Sigma–Aldrich, St Louis, MO), which contained 10% horse sera (Gibco, Carlsbad, CA) and passaged weekly on the basis of American Type Culture Collection recommendations. The implantation methods of sarcoma cells were previously described in our previous study.^18,19 Briefly, mice were anesthetized with an injection intraperitoneally of 50 mg/kg 1% pentobarbital sodium, and a superficial incision was conducted in the skin overlying the left patella, cutting the patellar ligament later and exposing the condyles of the distal femur. A 30-gauge needle was applied to perforate the bone cortex and a dental burr was used to get a 0.5-mm depression, then a 25 μl micro syringe was applied to implant a volume of 20 μl minimum essential medium (α-MEM) (sham-implanted mice) or 20 μl of medium containing 10⁵ NCTC 2472 cells (tumor-implanted mice) into the medullary cavity of the distal femora bone. Then, bone wax was used to seal the injection hole, followed by copious irrigation with naïve saline. Finally, the wound was ready to be closed. The following molecular biology and pathology experiments were conducted on day 14 and day 21 after injection in order to make the tumor fully occupies the distal femur by day 14 after tumor-implantation, and ensure the appearance of behaviors related to cancer-induced bone pain on day 10 after tumor-implantation and continue to escalate at day 17–21 after tumor-implantation when tumor had destroyed the fracture of the impaired femur.^19,20

Thermal hyperalgesia behavioral analysis

There was an experimenter, blind to the study, had detected all behavioral responses. The paw withdrawal thermal latency (PWL) to radiant thermal stimulation was applied to assessed the thermal hyperalgesia (Plantar Test 30370, Ugo Basile, Italy), according to our previous study. ¹⁹ Mice were raised in a clear plastic box and were allowed to habituate for 30 min. Then, PWL evoked by radiant threat were tested 3 times of each hind paw, with at least 5 min interval of each given paw. The mean PWL was assessed by the three stimuluses. Data were described as mean ± SD.

Histology and x-ray assessing the extent of bone destruction

CO2 asphyxiation was used to execute mice, and then bone tumor samples were extracted from the left femur, the samples should be mixed in 4% paraformaldehyde and prepared to pathological examination. The paraffin was used to embed the bones, and bones were separated into 4 μm sections (RM2016, Leica, Germany) and stained with standard hematoxylin and eosin (H&E) method to observe infiltration of tumor cell and femora destruction in tumor-implanted mice with a microscope (Nikon EclipseTI-SR, Leica, Japan). Standard x-ray film was applied to identify the extent of femora destruction (osteolysis) radiologically.

Quantitative RT-PCR (q RT-PCR) assay

The spinal MOR and KOR mRNA expression was measured by q RT-PCR. Total RNA was extracted with TRIzol reagent (Invitrogen, Carlsbad, CA, USA). A 2 μg of total RNA was employed for the synthesis of the first-strand cDNA with an M-MLV RT kit (Promega, Madison, WI USA). The PCR used to amply the resultant cDNA were the listed primers: MOR: 5'-TCTTCATCTTCGCCTTGA-3' (forward); and 5'-GGGTCCAGCAGACA ATAA-3'(reverse); KOR: 5’-TGGAGGCACCAAAGTCAG-3’ (forward) and 5’-TG GGATCAAAGGCAAA-3’ (reverse); β-actin: 5'-GTCCCTC ACCCTCCCAAAAG-3'(forward); and 5'-GCGCCTCAACACCTCAACCC-3' (reverse). PCR has conducted by the following thermal cycling conditions: denaturation at 95°C for 30 s followed by 40 cycles of denaturation at 95°C for 20 s, primer annealing at 55°C for 20 s, and primer extension at 72°C for 20 s, with a final extension at 72°C for 7 min on a Lightcycler 480 (Applied Biosystems, Foster City, CA, USA) using the SYBR Green Master Mix Kit (China). The expression level of MOR and KOR was normalized to that of the corresponding β-actin product followed by quantification using the 2^−ΔΔ Ct method.

Western blotting

The spinal MOR and KOR protein levels were determined by Western blotting. Following the manufacturer’s recommendations, the bicinchoninic acid assay (Keming Bioengineering Company, Suzhou, China) was applied to extract and quantify the total protein. A bicinchoninic acid (BCA) protein assay kit was used to measure the protein concentration. Samples were fixed with Laemmle sample buffer and boiled for 10 min. Then, a 30-μg protein sample was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, electro transferred to polyvinylidene fluoride membranes, blocked with 5% skim milk in Tris-buffered saline, and then immunoblotted with polyclonal anti-MOR (1:1,000), anti-KOR (1:1,000) and anti-actin antibody (1:1,000) overnight at 4°C. After rinsing, the membranes were incubated with horseradish peroxidase-conjugated anti-rabbit immunoglobulin at a dilution of 1:2000 for 1 h. Signals were visualized with enhanced chemiluminescence (ECL, Takara Bio, Japan) detection system. The films were observed and the band density was detected with NIH Image J software (National Institutes of Health; Bethesda, MD, USA). MOR or KOR protein levels were normalized to those of the corresponding β-actin product.

Animal treatment protocol and pharmacological testing

14 days after the inoculation of sarcoma cells, 24 BCP mice were successfully induced and randomly allocated into four groups using the random number table. Morphine and/or nalbuphine or saline was subcutaneously applied twice daily (at 7:30 a.m. and 7:30 p.m.) for seven consecutive days (from day 14 to day 20 after tumor implantation). There were four groups (n = 6 per group) as follows: the control group (1 ml isotonic saline, s. c.), Morphine group (10 mg/kg morphine, s. c.), Nalbuphine group (10 mg/kg nalbuphine, s. c.), and M + N group (10 mg/kg morphine +1 mg/kg nalbuphine, s. c.). The dosage of the drugs was determined by showing equipotent antinociceptive efficacy in various parameters after acute administration in mice. 30 min later, the potency of drugs were detected (subcutaneous, s. c.) at the timing of their peak effect. ²⁰ The average PWL of the behavioral test values in a day is compared among the groups.

Statistical analysis

Measurement data are listed in mean ± standard deviation (SD). GraphPad [version 8.4.2(679)] was used for data analysis. Behavioral measurement data was analyzed by one-way analysis of variance for repeated measures with Tukey’s multiple comparisons test. Other data was assessed by two-way ANOVA followed by Tukey’s multiple comparisons test. The level of significance was at 0.05. In this study, the investigator, was blind to plot, measurement, and count.

Results

Tumor-induced thermal hyperalgesia behavioral changes and bone destruction

Behaviorally, there was no statistical difference of the baseline (day 0) of PWL between sham-implanted mice and tumor-implanted mice. On day 4 and day 7 after procedure, both tumor-implanted and sham-implanted mice had descending PWL values (p < 0.05). However, the PWL of the sham-implanted mice began to return to the baseline on day 10 after surgery. On the contrary, tumor-implanted animals showed continuous alternation of thermal hyperalgesia behavior (Figure 2).

Figure 2.

Establishment of a bone cancer pain model. Paw withdrawal latency (PWL) to radiant heat progressively decreased as time went on in tumor-implanted mice. PWL values of sham-implanted mice began to recover on day 10 after surgery. Data were presented as the mean ± SD, and two-way analysis of variance (ANOVA) was used, followed by the Tukey’s multiple comparisons test. *p < 0.05 vs. sham-implanted mice. (n = 12 per group). (Interaction: F-statistic = 19.28; Row Factor: F-statistic = 40.66; Column Factor: F-statistic = 108.50).

As shown in Figure 3, the injection of sarcoma cells into the intramedullary canal of femur significantly performed bone destruction. Compared with sham-implanted mice (Figure 3(a)), on 21 d, Hematoxylin-eosin staining of tumor-implanted mice femora (Figure 3(b)) showed the displacement of the darkly stained marrow cells with the more lightly stained sarcoma cells had destroyed the bone and grown through the bone. The femur radiographs showed the progressive bone loss resulted from the tumor growth on 14 d (Figure 3, C-1) and 21 d (Figure 3, C-2).

Figure 3.

Pathological and radiological examination of ipsilateral bone tissues. Hematoxylin-eosin staining of 21 d sham-implanted mice femora (a) and that of tumor-implanted (b) mice, showing the replacement of the darkly stained marrow cells with the more lightly stained sarcoma cells that have induced the bone destruction and grown through the bone (arrow). Radiographs of the femur (c) show the progressive loss of bone caused by the tumor growth. Images 0–2 are examples of each state of destruction: 0, normal bone; 1, minor loss of bone in the medullary canal (arrow); 2, substantial loss of bone in the medullary canal with major structural destruction of the distal femur (arrow). Scale bars: A, B, 200 mm; C, 5 mm.

Tumor-induced changes in spinal MOR and KOR expression

It had proven that MOR and KOR are expressed in mice according to the relative quantification of MOR and KOR mRNA obtained from the spinal cord (L3–L5) from naïve animals. Compared with naïve mice, the expression of MOR (Figure 4) and KOR (Figure 4) mRNA of tumor-implanted mice gradually declined over time. On day 14 and day 21, there was a significant down-regulation of MOR and KOR mRNA in the spinal cord of tumor-implanted mice when compared with that of sham group and naïve group mice (p < 0.05). Western blotting analysis showed a single band respectively around 45 kDa (MOR) and 43 kDa (KOR) in samples extracted from the spinal cord (L3–L5) (Figures 5(a) and (b)). On days 14 and 21 after sarcoma inoculation, the protein level of MOR (Figures 5(c)) and KOR (Figures 5(d)) in the spinal cord was significantly decreased compared with those in sham-implanted and naïve group mice (p < 0.05).

Figure 4.

Quantitative Real-Time Reverse transcription-polymerase Chain Reaction analysis of changes of spinal MOR and KOR mRNA expression after the operation on mice. The spinal MOR and KOR mRNA expression progressively decreased as time went on in tumor-implanted mice. Data was presented as fold change of control (Naive) mean ± SD. *p < 0.05 vs. sham-implanted mice. (n = 4 per group). (Interaction: F-statistics = 3.39/16.91; Row Factor: F-statistics = 3.88/8.72; Column Factor: F-statistics = 116.40/231.80, MOR mRNA/KOR mRNA respectively).

Figure 5.

Changes of spinal MOR and KOR protein expression over time in tumor-implanted mice and sham-implanted mice. Western blot for β-actin, MOR, and KOR resulted in products of 42, 45, and 43 kDa, as expected (markers show predicted band sizes) (a and b). Densitometric quantification of β-actin, MOR, and KOR immunoreactivity on Western blots (c and d). Data were presented as fold change of control (Naive) mean ± SD.*p < 0.05 vs. sham-implanted mice. (n = 4 per group). (Interaction: F-statistics = 15.92/17.71; Row Factor: F-statistics = 43.83/25.90; Column Factor: F-statistics = 115.70/113.90, MOR protein/KOR protein respectively).

Effects of nalbuphine on morphine tolerance

We observed the PWL under thermal injury over 7 days, in order to assess the analgesic response of opioids in the BCP mice. At the beginning of the study, the overall mean baseline (day 0 as shown in Figure 6) of PWL to thermal injury was 6.87 ± 1.73 s, with no significant differences among all groups. Both the morphine group and nalbuphine group significantly delayed the PWL of the mice with BCP from days 1–4 compared with that of the sham group (p < 0.05). However, there were no significant differences between the morphine group and nalbuphine group from days 5–7, indicating that both morphine and nalbuphine can induce tolerance. Nalbuphine seemed to have a weaker analgesic effect compared with that of the morphine group, but the difference is not statistically significant. When co-administered with morphine, however, the application of low-dose nalbuphine (M + D group) significantly delayed the appearance of morphine tolerance. (Figure 6).

Figure 6.

Comparison of the paw withdrawal latency (PWL) among the groups. Twenty-four mice with bone cancer pain (BCP) were treated with opioids or normal saline twice daily for 7 days. The mice were randomly divided into four groups with different drug regimens (n = 6 per group): control group (1 ml isotonic saline, s. c.), Morphine group (10 mg/kg morphine, s. c.), Nalbuphine group (10 mg/kg nalbuphine, s. c.), and M + N group (10 mg/kg morphine +1 mg/kg nalbuphine, s. c.). All mice underwent thermal pain measurements 30 min after the drug administration at 7:30 a.m. and 7:30 p.m. . Data are expressed as the mean ± SEM, and two-way analysis of variance (ANOVA) was used, followed by Tukey’s multiple comparisons test for evaluation. *p < 0.05 compared with the control group. #p < 0.05 compared with the morphine group. (Interaction: F-statistic = 8.39; Row Factor: F-statistic = 36.29; Column Factor: F-statistic = 183.60).

Effects of opioids on spinal opioid receptors expression

At the levels of mRNA and protein expression, morphine therapy can lead to a decrease in spinal μ receptor protein and mRNA expression (p < 0.05), while only a decrease in kappa receptor protein expression (p < 0.05) without significantly affecting the expression of kappa receptor mRNA. Similarly, nalbuphine therapy can induce a decrease in the expression of κ receptor proteins and mRNA at the spinal cord level (p < 0.05), while only down-regulated the expression of μ receptor proteins (p < 0.05) without significantly changing the expression of μ receptor mRNA. A low dose of nalbuphine accompanied by morphine alleviated morphine-induced down-regulation of different types of opioid receptors (p < 0.05). (Figure 7).

Figure 7.

The mRNA and protein levels of opioid receptors in the spinal cord among the different groups. The groups were as follows (n = 6 per group): Control group (1 ml isotonic saline, s. c.), Morphine group (10 mg/kg morphine, s. c.), Nalbuphine group (10 mg/kg nalbuphine, s. c.), and M + N group (10 mg/kg morphine +1 mg/kg nalbuphine, s. c.). Data are expressed as the mean ± SEM. One-way analysis of variance (ANOVA) was used, followed by Tukey’s multiple comparisons test. *p < 0.05 compared with the control group. #p < 0.05 compared with the morphine group. (MOR mRNA: F-statistic = 144.70; KOR mRNA: F-statistic = 143.80; MOR protein: F-statistic = 28.54; KOR protein: F-statistics = 29.47).

Discussion

In this study, behavioral tests revealed that the inoculation of sarcoma cells into the left femur of male C3H/HeNCrlVr mice induced both progressive thermal hyperalgesia and femur destruction, suggesting that the model of BCP had been successfully established. These results were consistent with our previous study. ¹⁹ In our present study, the PWL decreased both in tumor-implanted and sham-implanted mice on day 4 and 7 after surgery, but showed no statistical difference among these groups at the two-time points. However, 10 days after surgery, the PWL value of sham group mice increased to the baseline, while that of tumor-implanted mice decreased further. These data implied that alternation of pain behavior on days 4 and 7 after surgery might attribute to gonarthrotomy but not only mainly resulting from the tumor.

MOR and KOR mRNA levels had appeared to have markedly decreased in the spinal cord in BCP mice. The mechanism of down-regulation expression of MOR and KOR may attribute to the reduction of the analgesic effects of opioids. We discovered that sarcoma injection itself can decrease the expression of MOR and KOR protein in the spinal cord, accompany the reduction of MOR and KOR mRNA expression. In peripheral nerve injury animal models, there was a down-regulation of MOR protein expression in the spinal cord.^21,22 Therefore, we could infer that the down-regulation of MOR expression in the spinal cord may suggest a nerve injury in BCP. Besides, we observed that the injection of sarcoma could decrease the expression of KOR in the spinal cord. The evidence implied that KOR might also matter in modulating BCP. However, the mechanism of why and how MOR and KOR expression decreased in BCP could not be inferred in our present study.

This study demonstrated that co-administration low dose of nalbuphine with morphine attenuated the appearance of morphine tolerance in a BCP mouse model. But the continuous application of conventional doses of morphine or nalbuphine has led to drug tolerance, manifested by a decrease in the analgesic effect. The dosage selection of drugs refers to the results of previous research and the conclusions of pre-experiments.^20,23,24 When morphine and nalbuphine had been combined, the analgesic effect from the MOR decreased, while the analgesic effect from the KOR increased. According to our results, the increased analgesic effects of the KOR can compensate the decreased analgesic effects of the MOR. We further explored the effects of opioid treatments on the expression of MOR and KOR in the spinal cord. We noticed that morphine therapy can lead to a decrease in spinal μ receptor protein and mRNA expression, while only a decrease in Kappa-receptor protein expression without significantly affecting the expression of Kappa-receptor mRNA. Similarly, nalbuphine therapy can decrease the expression of κ receptor proteins and mRNA at the spinal cord level (p < 0.05), while only down-regulating the expression of μ receptor proteins without significantly affecting the expression of μ receptor mRNA. There were three distinct processes that triggers changes of agonist activation in receptors' density and activity. Receptor desensitization is the alternation of the receptor function rather than receptor amount, while internalization means the movement of rapid agonist-induced receptor from the cell surface to the cytoplasm. Down-regulation means the decrease of total receptors because of less gene expression.^25,26,27 Morphine and nalbuphine have different effects on μ or κ receptors protein and mRNA expression may be due to the effects of different levels at the transcription level or translation stage respectively. However, the mechanism of function and expression changes of MOR and KOR during tumor development and opioid treatments still need to be further studied. Though traditional KOR agonists are generally owing to unexpected side effects, such as psychotomimesis, depression, anxiety, and dysphoria, the combining application of low-dose KOR agonists with MOR analgesy can decrease tolerance and further strengthen analgesia. Therefore, it is of equal significance to understand the changes in different opioid receptors caused by cancer pain itself and the changes in opioid receptor plasticity induced by opioid receptor agonists. In the next step, we will explore the mechanism of decreased opioid receptor expression induced by cancer pain itself and opioid receptor agonists.

Conclusion

In conclusion, BCP itself may induce down-regulation of MOR and KOR expression of C3H/HeNCrlVr Mice in the spinal cord level. Like MOR, KOR may also have potential in the regulation of cancer pain. A low dose of nalbuphine co-administration with morphine significantly had the emergence of morphine tolerance delayed and alleviated morphine-induced down-regulation of these MOR and KOR receptors.