**3. Treatment of bone cancer pain**

When a patient experiences bone cancer pain, the first step of therapy is tumor eradication, i.e. via chemotherapy and radiation, unfortunately in <50% of the patients the pain levels will return to pre-treatment levels [62]. Radiotherapy, described as the golden standard palliative therapy, shows full pain relief in 25% of treated patients, however, only after a month [29]. Different radiotherapy protocols showed a single radiotherapy fraction (8Gy) provides equal pain palliation compared to multiple fractions (30 or 20 Gy in 10 or 5 fractions, respectively) [63]. Low fractionated radiotherapy also caused a higher incidence of pathological fractions at site of irradiation [1]. Chemotherapy is an option for the treatment of CIBP when the tumor histology is more nociceptive, the patient did not previously receive chemotherapy and when the tumor is chemosensitive [64]. However, oxaliplatin and paclitaxel are used for animal models of induced-neuropathy to investigate hypersensitivity [65, 66].

#### **3.1 Bisphosphonates**

Bisphosphonates are agents that are often used to treat pain as a symptom [67]. They act by inhibiting farnesyl diphosphate synthase in phagocytic cells, e.g. osteoclasts, macrophages and microglia, thereby decrease extracellular acidification and consequently reduce ASIC- and TRPV1-mediated activation of nociceptive primary afferents located in bone [67]. Other effects of bisphosphonates unrelated to farnesyl diphosphate synthase inhibition that have been suggested are interactions with purinergic receptors, e.g. P2X7. The bisphosphonate zoledronate exerted an analgesic effects in rat CIBP models [68]. It is the most widely used bisphosphonate, also observed to significantly reduce CIBP in clinical practice for breast cancer metastases [69], being 100 to 1000 times more effective than pamidronate [70]. Furthermore, anti-inflammatory effects have been indicated where alendronate inhibited TNF-α, IL-1, IL-6 and NGF [67].

#### **3.2 Monoclonal antibody therapy**

Monoclonal antibody therapies have the ability to interfere with tumor-induced processes, e.g. RANK/RANKL, NGF/TrkA, and inhibit or avoid cytotoxic T

**9**

*Bone Cancer Pain, Mechanism and Treatment DOI: http://dx.doi.org/10.5772/intechopen.95910*

specifically targeting CIBP.

**3.3 Analgesics: NSAIDs and opioids**

moderate to severe pain (Step 3) [85].

lymphocyte [71]. A hand full of these therapies have been FDA approved for cancer therapy and a small amount has been tested in breast, prostate or lung cancer metastases [71]. Tanezumab is a monoclonal antibody interfering with NGF/TrkA and has been described unbeneficial in one CIBP study [72], however, has also been shown to attenuate late stage cancer pain [73]. Denosumab is another monoclonal antibody and acts by interfering with the interaction between RANK/ RANKL, capturing RANKL, resulting in osteoclast inactivation [74]. Denosumab has been tested as treatment in breast cancer metastases and while it showed a good activity profile for delaying or preventing skeletal related events, no direct relief of pain has been described. Nevertheless, the delay and/or prevention of skeletal related events would have an indirect pain-impairing potential as such events are associated with pain and increased morbidity [75]. Denosumab did show superiority concerning first on-study skeletal-related events compared to zoledronate [76]. Similar outcomes were found by a meta-analysis of 4 RCTs between denosumab and zoledronate [77]. Regarding the dosing, a study showed no difference between 4-weekly and 12-weekly administration for denosumab and the two bisphosphonates zoledronate and pamidronate, suggesting that incorporating 12-weekly dosing could benefit patients [78]. Denosumab seems to be the only antibody therapy so far that is approved for direct treatment of skeletalrelated events with bone metastases from solid tumors and giant cell tumors of the bone [71]. Ipilimumab is an antibody that activates the immune system, specifically, inhibits an inhibitory mechanism of cytotoxic T lymphocytes. It was tested in metastatic prostate cancer in combination with radiotherapy and suggested clinical antitumor activity [79]. Nivolumab therapy was recently tested in lung cancer metastases into the bone and showed that 40% of the treated patients had osteosclerotic change on CT scans, indicating successful treatment of bone lesions [80]. The small amount of monoclonal antibodies used for bone metastases often have skeletal related events as indication of efficacy but lack bone cancer pain as direct outcome measure. Currently there are no recorded monoclonal antibodies

Available options for the direct treatment of CIBP are analgesics. The WHO has established a 3-step ladder as a guideline for analgesic prescription in 1986 and revised the version in 1996 with a quick guide to opioid availability [81]. Afterwards, the stigma on opioid prescription was broken and received acceptance as treatment for (chronic) pain conditions [82–84]. The 3-step ladder starts with non-opioids (Step 1) for mild pain, weak opioids ± non-opioids and adjuvants for mild to moderate pain (Step 2), and strong opioids ± non-opioids and adjuvants for

First in line are NSAIDs that inhibit the enzyme cyclooxygenase-2 (COX-2), responsible for PGE synthesis [64]. A challenge with NSAIDs is that they reach a ceiling effect in analgesic efficacy [81, 86]. Increasing the doses does not result in increased efficacy, conversely, side effects worsen, further impairing the quality of life of patients [86, 87]. Second in line are weak opioids, e.g. codeine, tapentadol or tramadol, in combination with adjuvants, indicating proven analgesic efficacy in bone cancer pain [88]. There are three classical opioid receptors, e.g. the μ-, δ- and κ-opioid receptors (MOP, DOP and KOP receptor, respectively) and the later discovered Nocicpetin/OrphaninFQ opioid peptide (NOP) receptor [89]. These receptors are G-protein coupled receptors and upon activation initiate an intracellular cascade resulting in 1) the inhibition of adenylate cyclase (respon-

channels and

sible for cAMP production), 2) opening of inwardly rectifying K<sup>+</sup>

#### *Bone Cancer Pain, Mechanism and Treatment DOI: http://dx.doi.org/10.5772/intechopen.95910*

*Recent Advances in Bone Tumours and Osteoarthritis*

through A-δ and C-fibers was enhanced [61].

exact mechanism remains to be elucidated.

inhibited TNF-α, IL-1, IL-6 and NGF [67].

**3.2 Monoclonal antibody therapy**

**3. Treatment of bone cancer pain**

**3.1 Bisphosphonates**

and sprouting. The degradation of bone and the damage that occurs can activate mechanosensitive ion channels, e.g. TRPV, ASIC and P2X7 [29, 57, 58]. Activated NGF regulates the maintenance of the peripheral sensory neuron system and initiates sprouting of adjacent non-injured afferents upon injury or denervation, resulting in collateral sprouting [59, 60]. Random sprouting of sensory neurons co-expressing TrkA was shown in prostate cancer metastases [9, 48] and similar in breast cancer metastases [47]. Hypersensitivity occurs as a result of sprouting, causing sensitization of sensory nerves, which in its turn induces mechanonociception (by Aδ-fibers) [59]. Changes also have been shown to occur in the central nervous system in the spinal cord where the excitatory synaptic transmission mediated

On the one hand, it is suggested that the increase in activated osteoclasts causes the development of CIBP while on the other hand the secreted mediators directly exciting sensory nerve fibers is suggested to be the primary explanation [17, 51]. Nevertheless, all these multidisciplinary factors – *neurological, oncological and immunological* – contribute to CIBP and while they are described extensively, the

When a patient experiences bone cancer pain, the first step of therapy is tumor eradication, i.e. via chemotherapy and radiation, unfortunately in <50% of the patients the pain levels will return to pre-treatment levels [62]. Radiotherapy, described as the golden standard palliative therapy, shows full pain relief in 25% of treated patients, however, only after a month [29]. Different radiotherapy protocols showed a single radiotherapy fraction (8Gy) provides equal pain palliation compared to multiple fractions (30 or 20 Gy in 10 or 5 fractions, respectively) [63]. Low fractionated radiotherapy also caused a higher incidence of pathological fractions at site of irradiation [1]. Chemotherapy is an option for the treatment of CIBP when the tumor histology is more nociceptive, the patient did not previously receive chemotherapy and when the tumor is chemosensitive [64]. However, oxaliplatin and paclitaxel are used for animal models of induced-neuropathy to investigate hypersensitivity [65, 66].

Bisphosphonates are agents that are often used to treat pain as a symptom [67].

Monoclonal antibody therapies have the ability to interfere with tumor-induced

processes, e.g. RANK/RANKL, NGF/TrkA, and inhibit or avoid cytotoxic T

They act by inhibiting farnesyl diphosphate synthase in phagocytic cells, e.g. osteoclasts, macrophages and microglia, thereby decrease extracellular acidification and consequently reduce ASIC- and TRPV1-mediated activation of nociceptive primary afferents located in bone [67]. Other effects of bisphosphonates unrelated to farnesyl diphosphate synthase inhibition that have been suggested are interactions with purinergic receptors, e.g. P2X7. The bisphosphonate zoledronate exerted an analgesic effects in rat CIBP models [68]. It is the most widely used bisphosphonate, also observed to significantly reduce CIBP in clinical practice for breast cancer metastases [69], being 100 to 1000 times more effective than pamidronate [70]. Furthermore, anti-inflammatory effects have been indicated where alendronate

**8**

lymphocyte [71]. A hand full of these therapies have been FDA approved for cancer therapy and a small amount has been tested in breast, prostate or lung cancer metastases [71]. Tanezumab is a monoclonal antibody interfering with NGF/TrkA and has been described unbeneficial in one CIBP study [72], however, has also been shown to attenuate late stage cancer pain [73]. Denosumab is another monoclonal antibody and acts by interfering with the interaction between RANK/ RANKL, capturing RANKL, resulting in osteoclast inactivation [74]. Denosumab has been tested as treatment in breast cancer metastases and while it showed a good activity profile for delaying or preventing skeletal related events, no direct relief of pain has been described. Nevertheless, the delay and/or prevention of skeletal related events would have an indirect pain-impairing potential as such events are associated with pain and increased morbidity [75]. Denosumab did show superiority concerning first on-study skeletal-related events compared to zoledronate [76]. Similar outcomes were found by a meta-analysis of 4 RCTs between denosumab and zoledronate [77]. Regarding the dosing, a study showed no difference between 4-weekly and 12-weekly administration for denosumab and the two bisphosphonates zoledronate and pamidronate, suggesting that incorporating 12-weekly dosing could benefit patients [78]. Denosumab seems to be the only antibody therapy so far that is approved for direct treatment of skeletalrelated events with bone metastases from solid tumors and giant cell tumors of the bone [71]. Ipilimumab is an antibody that activates the immune system, specifically, inhibits an inhibitory mechanism of cytotoxic T lymphocytes. It was tested in metastatic prostate cancer in combination with radiotherapy and suggested clinical antitumor activity [79]. Nivolumab therapy was recently tested in lung cancer metastases into the bone and showed that 40% of the treated patients had osteosclerotic change on CT scans, indicating successful treatment of bone lesions [80]. The small amount of monoclonal antibodies used for bone metastases often have skeletal related events as indication of efficacy but lack bone cancer pain as direct outcome measure. Currently there are no recorded monoclonal antibodies specifically targeting CIBP.

### **3.3 Analgesics: NSAIDs and opioids**

Available options for the direct treatment of CIBP are analgesics. The WHO has established a 3-step ladder as a guideline for analgesic prescription in 1986 and revised the version in 1996 with a quick guide to opioid availability [81]. Afterwards, the stigma on opioid prescription was broken and received acceptance as treatment for (chronic) pain conditions [82–84]. The 3-step ladder starts with non-opioids (Step 1) for mild pain, weak opioids ± non-opioids and adjuvants for mild to moderate pain (Step 2), and strong opioids ± non-opioids and adjuvants for moderate to severe pain (Step 3) [85].

First in line are NSAIDs that inhibit the enzyme cyclooxygenase-2 (COX-2), responsible for PGE synthesis [64]. A challenge with NSAIDs is that they reach a ceiling effect in analgesic efficacy [81, 86]. Increasing the doses does not result in increased efficacy, conversely, side effects worsen, further impairing the quality of life of patients [86, 87]. Second in line are weak opioids, e.g. codeine, tapentadol or tramadol, in combination with adjuvants, indicating proven analgesic efficacy in bone cancer pain [88]. There are three classical opioid receptors, e.g. the μ-, δ- and κ-opioid receptors (MOP, DOP and KOP receptor, respectively) and the later discovered Nocicpetin/OrphaninFQ opioid peptide (NOP) receptor [89]. These receptors are G-protein coupled receptors and upon activation initiate an intracellular cascade resulting in 1) the inhibition of adenylate cyclase (responsible for cAMP production), 2) opening of inwardly rectifying K<sup>+</sup> channels and

3) closing of voltage-gated Ca2+ channels [89]. Caution must be exercised with weak opioids as the rate of metabolism by Cytochrome P450 enzymes defines analgesic efficacy and side effects. In addition, codeine seemed effective for only 1 month until strong opioids were necessary for adequate analgesia [90, 91]. A randomized RCT trial showed significant impairment of cancer pain by low-dose morphine compared with weak opioids, with similar tolerability and an earlier effect, suggesting low-dose morphine can be used [90, 92]. This forwards the therapy option towards Step 3 and to date, the first choice to treat moderate to severe pain with strong opioids remains morphine [90, 93]. MOP receptor drugs have shown superior analgesic efficacy and have been used for centuries as they seem to be the most potent analgesics [94]. Available options for administration are oral and transdermal, showing similar efficacy, and advocated is the use of epidural or intrathecal pumps if relief is inadequate [90]. Concerning side effects of MOP receptor drugs are addiction and dependency. The opioid crisis is prove and accounted for 33.000 deaths per year in the US by opioid misuse [94–96]. In addition, cancer survivors showed higher opioid prescription compared to controls [97]. The total estimated economic burden due to opioid addiction, dependency, abuse and overdose is \$78.5 billion, from which \$28.9 billion is due to increased health care and abuse treatment [98]. Furthermore, analgesic efficacy of MOP receptor compounds is affected by long term opioid treatment as tolerance develops over time [99, 100]. This is inevitable in cancer patients since high doses are required for pain management [101]. The mechanism that contributes pre-synaptically to tolerance remains to be elucidated but TRPV1 receptor upregulation in spinal cord and dorsal root ganglions has been shown to accompany tolerance [99, 100].

Challenging is to find analgesics with a similar potency and efficacy compared to MOP receptors, without dependency and addiction. Targeting the DOP and KOP receptor showed efficacious pain relief with a lower abuse potential, making them promising targets for treating pain [102]. Specifically for CIBP, both DOP and KOP receptor agonists showed pain attenuation in animal models of CIBP [103, 104]. It has been shown that a selective KOP receptor agonist blocked pain without altering bone loss, tumor size or cancer cell proliferation [105]. Additionally, a DOP receptor agonist showed equal analgesic efficacy and 4.5-fold potency compared to morphine in a mouse CIBP model [106]. Despite potential analgesic efficacy, MOP receptor agonists remain the clinical mainstay [107, 108]. Interest in the NOP receptor increased after the discovery of similar, yet distinct features compared to the classical opioids [109]. The effects of classical opioids are immediately blocked by naloxone and independently of administration location, they attenuate pain. The analgesic NOP receptor effect remains after naloxone and interestingly, spinal or peripheral activation exerts anti-nociceptive effects, while supra-spinally it acts pro-nociceptive [85, 109]. Following these discoveries, the NOP receptor showed anti-rewarding and anti-abuse effects in rodents [85, 110–113]. Furthermore, NOP receptor expressing Chinese Hamster Ovary cells showed rapid internalization after activation and a quick recycle process to reactivate receptors occurred at the membrane, potentially reducing the development of tolerance. However, compensatory mechanisms that remain to be elucidated may be overlooked [114]. The NOP receptor has been specifically used to target CIBP and both the endogenous ligand Nociceptin and a synthetic selective NOP receptor agonist (Ro65–6570) showed significant analgesia [85]. Furthermore, NOP receptor activation downregulates IL-6 production [115] and is suggested to inhibit T cell proliferation [116]. Altogether, the anti-rewarding and anti-abuse effects, cytokine production involvement and selective attenuation of CIBP, makes the NOP receptor an interesting target.

**11**

*Bone Cancer Pain, Mechanism and Treatment DOI: http://dx.doi.org/10.5772/intechopen.95910*

**3.4 Primary vs. secondary tumor treatment**

address their metastatic counterparts [119].

**4. Bone cancer pain: research techniques**

**4.1** *In Vivo* **models for bone cancer pain**

**3.5 Non-pharmacological interventional treatment**

alcohol), 6% aqueous phenol and 6% phenol in glycerine [120].

Differences should be kept in mind when treating tumors, nevertheless, anti-NGF antibody therapy has been observed to relief early and late stage CIBP in a primary bone tumor model and a metastatic-like prostate bone cancer model [37]. In addition, zoledronate has been shown effective in reducing the risk of skeletal related events in multiple myeloma, prostate and breast cancer bone metastasis [117]. Denosumab indicated superiority to zoledronate in preventing skeletal related events in bone metastasis compared to solid tumors, suggesting a treatment option for bone metastasis [118]. Primary bone tumors are characterized by high complexity and heterogeneity in genomic alterations and are therefore challenging for developing targeted therapeutic strategies [41] which also may not satisfactorily

The WHO analgesic ladder has proven to be very helpful, nevertheless, an estimated 12% of patients remains inadequately treated for CIBP [120]. Therefore, a fourth step has been proposed that includes interventional approaches to provide a minimal acceptable quality of life [120–122]. As such, percutaneous neurolysis is performed using chemical agents or thermal energy upon celiac plexus, splanchnic nerve, superior hypogastric plexus, brachial plexus and epidural and intrathecal [120, 122]. Commonly used neurolytic agents are absolute alcohol (diluted to 50%

Finally, PET/CT allows the distinction between osteolytic and osteoblastic lesions and thereby detect more subtle responses to treatment regimens [123]. Using CT in the surgical planning could shift the priority of debulking dense bone to surgical reconstruction when bone metastasis is more osteolytic instead of osteoblastic [39].

The current treatments are often targeted against pain as a symptom and therapy options specifically for CIBP are rare. To elucidate the complex mechanism of action of CIBP and develop novel analgesics, further research is warranted. As such, *in vitro* techniques are an option, however, these capture a minor aspect of the complexity and as long as no technique exists that simulates this, *in vivo* research is inevitable. Nevertheless, it should be conducted highly ethically and additional regulations were established in 2009 to maintain the animals' welfare by following 3R's (Reduction, Refinement and Replacement) [124]. Furthermore, to test a nociceptive phenotype in a comfortable manner, more focus is towards animals' ethological and evolutionary preserved behavior. Finally, the *in vivo* model that is used should represent the disease and clinical symptoms as close as possible. Three criteria are important in the validation of animal models [125], 1) Face validity: the biology and symptoms as seen in humans are similar in the animal model, 2) Predictive validity: if the clinical intervention has an equal response in the animal model and 3) Construct validity: the target one is investigating exerts the same biological processes in both organisms, e.g. opioid receptors are responsible for pain relief.

At start, to reflect metastases as closely as possible, cancer cells were injected either intravenously or intra-cardially. Face validity is achieved but uncontrolled *Recent Advances in Bone Tumours and Osteoarthritis*

3) closing of voltage-gated Ca2+ channels [89]. Caution must be exercised with weak opioids as the rate of metabolism by Cytochrome P450 enzymes defines analgesic efficacy and side effects. In addition, codeine seemed effective for only 1 month until strong opioids were necessary for adequate analgesia [90, 91]. A randomized RCT trial showed significant impairment of cancer pain by low-dose morphine compared with weak opioids, with similar tolerability and an earlier effect, suggesting low-dose morphine can be used [90, 92]. This forwards the therapy option towards Step 3 and to date, the first choice to treat moderate to severe pain with strong opioids remains morphine [90, 93]. MOP receptor drugs have shown superior analgesic efficacy and have been used for centuries as they seem to be the most potent analgesics [94]. Available options for administration are oral and transdermal, showing similar efficacy, and advocated is the use of epidural or intrathecal pumps if relief is inadequate [90]. Concerning side effects of MOP receptor drugs are addiction and dependency. The opioid crisis is prove and accounted for 33.000 deaths per year in the US by opioid misuse [94–96]. In addition, cancer survivors showed higher opioid prescription compared to controls [97]. The total estimated economic burden due to opioid addiction, dependency, abuse and overdose is \$78.5 billion, from which \$28.9 billion is due to increased health care and abuse treatment [98]. Furthermore, analgesic efficacy of MOP receptor compounds is affected by long term opioid treatment as tolerance develops over time [99, 100]. This is inevitable in cancer patients since high doses are required for pain management [101]. The mechanism that contributes pre-synaptically to tolerance remains to be elucidated but TRPV1 receptor upregulation in spinal cord and dorsal root ganglions has been shown to accompany

Challenging is to find analgesics with a similar potency and efficacy compared to MOP receptors, without dependency and addiction. Targeting the DOP and KOP receptor showed efficacious pain relief with a lower abuse potential, making them promising targets for treating pain [102]. Specifically for CIBP, both DOP and KOP receptor agonists showed pain attenuation in animal models of CIBP [103, 104]. It has been shown that a selective KOP receptor agonist blocked pain without altering bone loss, tumor size or cancer cell proliferation [105]. Additionally, a DOP receptor agonist showed equal analgesic efficacy and 4.5-fold potency compared to morphine in a mouse CIBP model [106]. Despite potential analgesic efficacy, MOP receptor agonists remain the clinical mainstay [107, 108]. Interest in the NOP receptor increased after the discovery of similar, yet distinct features compared to the classical opioids [109]. The effects of classical opioids are immediately blocked by naloxone and independently of administration location, they attenuate pain. The analgesic NOP receptor effect remains after naloxone and interestingly, spinal or peripheral activation exerts anti-nociceptive effects, while supra-spinally it acts pro-nociceptive [85, 109]. Following these discoveries, the NOP receptor showed anti-rewarding and anti-abuse effects in rodents [85, 110–113]. Furthermore, NOP receptor expressing Chinese Hamster Ovary cells showed rapid internalization after activation and a quick recycle process to reactivate receptors occurred at the membrane, potentially reducing the development of tolerance. However, compensatory mechanisms that remain to be elucidated may be overlooked [114]. The NOP receptor has been specifically used to target CIBP and both the endogenous ligand Nociceptin and a synthetic selective NOP receptor agonist (Ro65–6570) showed significant analgesia [85]. Furthermore, NOP receptor activation downregulates IL-6 production [115] and is suggested to inhibit T cell proliferation [116]. Altogether, the anti-rewarding and anti-abuse effects, cytokine production involvement and selective attenuation of CIBP, makes the NOP receptor an

**10**

interesting target.

tolerance [99, 100].

## **3.4 Primary vs. secondary tumor treatment**

Differences should be kept in mind when treating tumors, nevertheless, anti-NGF antibody therapy has been observed to relief early and late stage CIBP in a primary bone tumor model and a metastatic-like prostate bone cancer model [37]. In addition, zoledronate has been shown effective in reducing the risk of skeletal related events in multiple myeloma, prostate and breast cancer bone metastasis [117]. Denosumab indicated superiority to zoledronate in preventing skeletal related events in bone metastasis compared to solid tumors, suggesting a treatment option for bone metastasis [118]. Primary bone tumors are characterized by high complexity and heterogeneity in genomic alterations and are therefore challenging for developing targeted therapeutic strategies [41] which also may not satisfactorily address their metastatic counterparts [119].

## **3.5 Non-pharmacological interventional treatment**

The WHO analgesic ladder has proven to be very helpful, nevertheless, an estimated 12% of patients remains inadequately treated for CIBP [120]. Therefore, a fourth step has been proposed that includes interventional approaches to provide a minimal acceptable quality of life [120–122]. As such, percutaneous neurolysis is performed using chemical agents or thermal energy upon celiac plexus, splanchnic nerve, superior hypogastric plexus, brachial plexus and epidural and intrathecal [120, 122]. Commonly used neurolytic agents are absolute alcohol (diluted to 50% alcohol), 6% aqueous phenol and 6% phenol in glycerine [120].

Finally, PET/CT allows the distinction between osteolytic and osteoblastic lesions and thereby detect more subtle responses to treatment regimens [123]. Using CT in the surgical planning could shift the priority of debulking dense bone to surgical reconstruction when bone metastasis is more osteolytic instead of osteoblastic [39].

## **4. Bone cancer pain: research techniques**

The current treatments are often targeted against pain as a symptom and therapy options specifically for CIBP are rare. To elucidate the complex mechanism of action of CIBP and develop novel analgesics, further research is warranted. As such, *in vitro* techniques are an option, however, these capture a minor aspect of the complexity and as long as no technique exists that simulates this, *in vivo* research is inevitable. Nevertheless, it should be conducted highly ethically and additional regulations were established in 2009 to maintain the animals' welfare by following 3R's (Reduction, Refinement and Replacement) [124]. Furthermore, to test a nociceptive phenotype in a comfortable manner, more focus is towards animals' ethological and evolutionary preserved behavior. Finally, the *in vivo* model that is used should represent the disease and clinical symptoms as close as possible. Three criteria are important in the validation of animal models [125], 1) Face validity: the biology and symptoms as seen in humans are similar in the animal model, 2) Predictive validity: if the clinical intervention has an equal response in the animal model and 3) Construct validity: the target one is investigating exerts the same biological processes in both organisms, e.g. opioid receptors are responsible for pain relief.

#### **4.1** *In Vivo* **models for bone cancer pain**

At start, to reflect metastases as closely as possible, cancer cells were injected either intravenously or intra-cardially. Face validity is achieved but uncontrolled

#### **Figure 2.**

*A representation of the different in vivo models to study cancer induced bone pain.*

growth of tumors occurs [32, 126, 127]. Next came the technique of injecting osteosarcoma-derived mesenchymal cells (NCTC-2472) directly into the long bones of mice [128]. This technique indicates good face and predictive validity, resulting in a controlled late-phase CIBP model, reflecting the clinical course with a comparable responsiveness to systemic opioid treatment [32, 128]. Finally, construct validity had been optimized using syngeneic cell lines (originating from the same species). The first example was rat mammary gland carcinoma cells (MRMT-1 cell line) inoculation into the tibia of rats [129]. The main characteristics after inoculation of cancer cells are: development of allodynia and hyperalgesia, progressive tumor growth, profound destruction and rebuilding of bone and no external tumor growth into other organs. In addition, upregulation of TNF-α, Interferon- γ (IFN-γ), IL-1β, IL-4, IL-10 and IL-6 occurs in tumor-bearing animals [49, 130]. Fine-tuning occurred with another rat breast cancer cell line (Walker 256 cells) inoculated into the tibia [131]. This model has been reviewed extensively and develops spontaneous pain, hyperalgesia, allodynia as well as ambulatory pain, indicates progressive tumor growth with osteolysis and no external growth, including upregulation of IL-1β, NGF, PGE2, IL-6 and TNFα [132]. This model has been subjected to a detailed pharmacological profiling using standard analgesic drugs for CIBP in a clinical setting and is suggested to be one of the most suitable preclinical models for novel compound identification and assessment [132, 133]. No study has been conducted comparing the Walker 256 model with the MRMT-1 model (**Figure 2**).
