*2.5. Sodium Dodecyl Sulfate–Polyacrylamide Gel Electrophoresis (SDS-PAGE)*

The control and resulting acetylated RPC were analyzed by SDS-PAGE (Figure 5). Based on the electrophoretic analyses, it was revealed that the rice protein not subjected to acetylation was composed of glutelin, with a molecular mass of about 22 kDa and 35–39 kDa, and prolamin, with a molecular mass of about 18 kDa. Similar observations were presented by Wang et al. [11], who additionally observed globulin and proglutelin fractions with molecular masses of 26 kDa and about 57 kDa, respectively. Based on the data in the literature, the major component of rice protein is glutelin, constituting about 80% of the total endosperm protein: prolamin, a minor storage protein, accounts for 5% to 10%. Other proteins include fractions such as globulin and albumin [11].

Based on the presented studies, it was shown that with an increase in the concentration of the modifying reagent (from 0.4 to 2.0 mL/g), the content of the main rice protein fractions, i.e., prolamin and glutelin, decreased. The applied acetylation process contributed to the removal of those proteins with a mass of about 18 kDa and about 35 kDa. This also confirmed a decrease in the protein content of the acetylated preparations. Similar results were observed in previous studies [28] when pumpkin protein concentrate was subjected to the same chemical modification.


**5. Enhancement of efficacy**

A major concern in the treatment of prostate cancer patients in the more advanced stage of the disease is the delicate balance between efficacy and toxicity. Treatment of metastatic bone pain with analgesics or localized external beam radiotherapy is relatively safe and easy. Treatment with bone seeking radiopharmaceuticals may be more appropriate in selected cases but efficacy is sometimes disappointing and bone marrow toxicity may be high in individual patients. Enhancement of overall efficacy without increasing toxicity could push the clinical decision algorithm in a positive direction with regard to the use of bone seeking radiopharmaceuticals.

One way of improving overall efficacy is combined treatment. Combined treatment regimens may deliver the beneficial effect of two different treatment modalities. These

Bone Seeking Radiopharmaceuticals for Metastatic Bone Pain 227

89Sr-Chloride to chemotherapy may lead to improved survival and a longer duration of the pain response compared to treatment with chemotherapy alone (Tu et al. 2001). Studies are yet limited but they are encouraging. Most of them have been performed using 89Sr-Chloride and some using 153Sm-EDTMP (Ricci et al. 2007). Patients may possibly have an improved pain response and longer survival. In prostate cancer patients a most interesting choice would be combining docetaxel and a bone seeking radiopharmaceutical. These studies, using 153Sm-EDTMP or 186Re-HEDP, are underway. Our group investigated the combination of 188Re-HEDP and capecitabine (Xeloda®) (Lam et al. 2009a). This treatment regimen proved to be feasible and safe. Phase II efficacy testing using the maximum tolerable dose of 2500 mg/m2/day capecitabine is underway. Capecitabine is primarily used as a radiation sensitizer. It offers a convenient therapy with acceptable toxicity, ease of use as oral tablets and low costs. The same is true for 188Re-HEDP. It is homemade on demand and has favourable physical characteristics with a short half-life (16.9 hours) and high betaenergy (Eβ max 2.12 MeV; Eβ mean 0.76 MeV). In theory, this high dose rate could lead to improved efficacy with rapid recovery. This is ideal for combined treatment regimens and for repeated treatment, which has already shown to be favourable with regard to pain

Several other combinations using bone seeking radiopharmaceuticals are under investigation. Currently, a randomized phase III study (MDA-3410/CTSU in M.D. Anderson Cancer Center, Houston, Texas) combining weekly doxorubicin (20 mg/m2) with 89Sr-Chloride after response to induction chemotherapy is underway, as well as another phase I trial combining docetaxel/prednisone and 153Sm-EDTMP, also at M.D. Anderson (Tu and Lin 2008). It remains to be established whether targeting both the tumor (chemotherapy) and bone compartments will improve therapeutic efficacy. This concept is also being tested in multiple other trials, mostly combining docetaxel/prednisone with bone environment directed treatments, like the agents described above (Tu and Lin 2008). The described studies add to this search for optimized treatment regimens in hormone-

Most patients with advanced prostate cancer have disease limited to the bone, which is notoriously difficult to assess for response, with a small subset having soft tissue lesions. To limit response evaluation to only patients with bidimensionally measurable disease would eliminate 70% to 80% of patients who would otherwise be evaluable (Figg et al. 1996). With regard to prostate cancer patients efficacy of treatment has been monitored by PSA in a majority of studies. However it is doubtful whether PSA changes correlate with clinical benefit (Bubley et al. 1999). A 50% decrease in PSA level seems a reasonable predictor of a favourable outcome, but this was certainly not the case in all studies (Bauer et al. 1999; Sridhara et al. 1995). It was advised not to use PSA level drops as a surrogate marker for

In patients with advanced disease, survival and quality of life, including pain palliation, are the most important criteria of clinical response. Besides survival these parameters are difficult to measure and subject to errors. With an attempt to standardize treatment response monitoring in cancer patients the European Organization for Research and Treatment of Cancer (EORTC) has developed quality of life questionnaires (EORTC QLQ-C30 version 3.0). They were validated in many clinical trials (Aaronson et al. 1993). They

response and survival (Palmedo et al. 2003).

refractory prostate cancer patients.

survival (Bubley et al. 1999).

**5.2 Predictions of efficacy and toxicity**

combinations may not only be additive but possibly synergistic to each other, leading to enhancement of overall efficacy with acceptable toxicity.

#### **5.1 Multimodality treatment**

The propensity of prostate cancer to metastasize to bone and the prognostic significance of bone metastases suggest that effective treatment of bone metastases may provide clinical benefits (DePuy et al. 2007; Sabbatini et al. 1999). With regard to the 'seed' and 'soil' theory on bone metastases the seed may comprise the so-called cancer stem cells. Whereas the soil may comprise a unique microenvironment, that facilitate the growth and survival of cancer stem cells. Targeting the microenvironment may offer another way to improve treatment of prostate cancer bone metastases. The microenvironment consists of osteoclasts, osteoblasts, endothelium and stroma. In the presence of cancer stem cells they interact leading to a disruption in normal coupling between osteoclasts and osteoblasts. An improved understanding of this process will influence how we select agents to target bone metastases and how we design strategies to treat prostate cancer bone metastases. Treatments may be directed to the cancer stem cells, the osteoblasts, the osteoclasts, the endothelium or the stroma (Tu and Lin 2008).

Osteoblasts may be targeted by several pharmaceuticals including bone seeking radiopharmaceuticals. Other osteoblast directed treatments include endothelin-1 antagonists (atrasentan) (Carducci et al. 2003), vitamin D analogs (1,25-hydroxyvitamin D3) (Beer et al. 2007), monoclonal IGF-1R (insulin-like growth factor-1-receptor) antibodies (Boyle et al. 2001) and CXRC4 (G-protein-coupled receptor) inhibitors (MSX-122), which inhibit the homing behaviour of cancer stem cells. Osteoclast activity may be inhibited by bisphosphonates (zolendronate) (Saad et al. 2002), RANK ligand inhibitors (denosumab) (Lewiecki 2006), tyrosine kinase inhibitors (dasatinib) or IL-6 antagonists (CNTO328) (Nam et al. 2005). And the endothelium and/or stroma may be targeted by anti-angiogenesis therapies. These include the vascular endothelial growth factor (VEGF) receptor antagonists bevacizumab, thalidomide and lenalidomide (Dahut et al. 2004). They reduce VEGF levels and basic fibroblast growth factor, inhibit growth and survival of tumor cells by modulation of adhesion molecules and mediate various cytokines. Also targeted to the endothelium are platelet-derived growth factor receptor (PDGFR) tyrosine kinase inhibitors (imatinib, sunitinib and tandutinib) (Ko et al. 2001). They may also have anti-angiogenetic potential. Most of these agents are under investigation. Many clinical studies in prostate cancer patients are ongoing (Tu and Lin 2008).

Combining different treatment modalities may be interesting because of additive effects or synergy effects. In the case of bisphosphonates and bone seeking radiopharmaceuticals the combined use has always been contra-indicated because of presumed interaction at the bone matrix level. It was shown that the combined use of 153Sm-EDTMP and bisphosphonates in patients with hormone-refractory prostate carcinoma is feasible. The combined treatment regimen is safe and may prove to be an effective long-term treatment regimen (Lam et al. 2007).

Another combined treatment regimen would be a combination of bone seeking radiopharmaceuticals and chemotherapy. It is likely that combined treatment with 89Sr-Chloride and chemotherapy (platinum based) yields a better pain response than treatment without chemotherapy in patients with painful osseous metastases from a prostate carcinoma (Sciuto et al. 2002), (Sciuto et al. 1996). And it has been suggested that adding

combinations may not only be additive but possibly synergistic to each other, leading to

The propensity of prostate cancer to metastasize to bone and the prognostic significance of bone metastases suggest that effective treatment of bone metastases may provide clinical benefits (DePuy et al. 2007; Sabbatini et al. 1999). With regard to the 'seed' and 'soil' theory on bone metastases the seed may comprise the so-called cancer stem cells. Whereas the soil may comprise a unique microenvironment, that facilitate the growth and survival of cancer stem cells. Targeting the microenvironment may offer another way to improve treatment of prostate cancer bone metastases. The microenvironment consists of osteoclasts, osteoblasts, endothelium and stroma. In the presence of cancer stem cells they interact leading to a disruption in normal coupling between osteoclasts and osteoblasts. An improved understanding of this process will influence how we select agents to target bone metastases and how we design strategies to treat prostate cancer bone metastases. Treatments may be directed to the cancer stem cells, the osteoblasts, the osteoclasts, the endothelium or the

Osteoblasts may be targeted by several pharmaceuticals including bone seeking radiopharmaceuticals. Other osteoblast directed treatments include endothelin-1 antagonists (atrasentan) (Carducci et al. 2003), vitamin D analogs (1,25-hydroxyvitamin D3) (Beer et al. 2007), monoclonal IGF-1R (insulin-like growth factor-1-receptor) antibodies (Boyle et al. 2001) and CXRC4 (G-protein-coupled receptor) inhibitors (MSX-122), which inhibit the homing behaviour of cancer stem cells. Osteoclast activity may be inhibited by bisphosphonates (zolendronate) (Saad et al. 2002), RANK ligand inhibitors (denosumab) (Lewiecki 2006), tyrosine kinase inhibitors (dasatinib) or IL-6 antagonists (CNTO328) (Nam et al. 2005). And the endothelium and/or stroma may be targeted by anti-angiogenesis therapies. These include the vascular endothelial growth factor (VEGF) receptor antagonists bevacizumab, thalidomide and lenalidomide (Dahut et al. 2004). They reduce VEGF levels and basic fibroblast growth factor, inhibit growth and survival of tumor cells by modulation of adhesion molecules and mediate various cytokines. Also targeted to the endothelium are platelet-derived growth factor receptor (PDGFR) tyrosine kinase inhibitors (imatinib, sunitinib and tandutinib) (Ko et al. 2001). They may also have anti-angiogenetic potential. Most of these agents are under investigation. Many clinical studies in prostate cancer

Combining different treatment modalities may be interesting because of additive effects or synergy effects. In the case of bisphosphonates and bone seeking radiopharmaceuticals the combined use has always been contra-indicated because of presumed interaction at the bone matrix level. It was shown that the combined use of 153Sm-EDTMP and bisphosphonates in patients with hormone-refractory prostate carcinoma is feasible. The combined treatment regimen is safe and may prove to be an effective long-term treatment

Another combined treatment regimen would be a combination of bone seeking radiopharmaceuticals and chemotherapy. It is likely that combined treatment with 89Sr-Chloride and chemotherapy (platinum based) yields a better pain response than treatment without chemotherapy in patients with painful osseous metastases from a prostate carcinoma (Sciuto et al. 2002), (Sciuto et al. 1996). And it has been suggested that adding

enhancement of overall efficacy with acceptable toxicity.

**5.1 Multimodality treatment**

stroma (Tu and Lin 2008).

patients are ongoing (Tu and Lin 2008).

regimen (Lam et al. 2007).

89Sr-Chloride to chemotherapy may lead to improved survival and a longer duration of the pain response compared to treatment with chemotherapy alone (Tu et al. 2001). Studies are yet limited but they are encouraging. Most of them have been performed using 89Sr-Chloride and some using 153Sm-EDTMP (Ricci et al. 2007). Patients may possibly have an improved pain response and longer survival. In prostate cancer patients a most interesting choice would be combining docetaxel and a bone seeking radiopharmaceutical. These studies, using 153Sm-EDTMP or 186Re-HEDP, are underway. Our group investigated the combination of 188Re-HEDP and capecitabine (Xeloda®) (Lam et al. 2009a). This treatment regimen proved to be feasible and safe. Phase II efficacy testing using the maximum tolerable dose of 2500 mg/m2/day capecitabine is underway. Capecitabine is primarily used as a radiation sensitizer. It offers a convenient therapy with acceptable toxicity, ease of use as oral tablets and low costs. The same is true for 188Re-HEDP. It is homemade on demand and has favourable physical characteristics with a short half-life (16.9 hours) and high betaenergy (Eβ max 2.12 MeV; Eβ mean 0.76 MeV). In theory, this high dose rate could lead to improved efficacy with rapid recovery. This is ideal for combined treatment regimens and for repeated treatment, which has already shown to be favourable with regard to pain response and survival (Palmedo et al. 2003).

Several other combinations using bone seeking radiopharmaceuticals are under investigation. Currently, a randomized phase III study (MDA-3410/CTSU in M.D. Anderson Cancer Center, Houston, Texas) combining weekly doxorubicin (20 mg/m2) with 89Sr-Chloride after response to induction chemotherapy is underway, as well as another phase I trial combining docetaxel/prednisone and 153Sm-EDTMP, also at M.D. Anderson (Tu and Lin 2008). It remains to be established whether targeting both the tumor (chemotherapy) and bone compartments will improve therapeutic efficacy. This concept is also being tested in multiple other trials, mostly combining docetaxel/prednisone with bone environment directed treatments, like the agents described above (Tu and Lin 2008). The described studies add to this search for optimized treatment regimens in hormonerefractory prostate cancer patients.

#### **5.2 Predictions of efficacy and toxicity**

Most patients with advanced prostate cancer have disease limited to the bone, which is notoriously difficult to assess for response, with a small subset having soft tissue lesions. To limit response evaluation to only patients with bidimensionally measurable disease would eliminate 70% to 80% of patients who would otherwise be evaluable (Figg et al. 1996). With regard to prostate cancer patients efficacy of treatment has been monitored by PSA in a majority of studies. However it is doubtful whether PSA changes correlate with clinical benefit (Bubley et al. 1999). A 50% decrease in PSA level seems a reasonable predictor of a favourable outcome, but this was certainly not the case in all studies (Bauer et al. 1999; Sridhara et al. 1995). It was advised not to use PSA level drops as a surrogate marker for survival (Bubley et al. 1999).

In patients with advanced disease, survival and quality of life, including pain palliation, are the most important criteria of clinical response. Besides survival these parameters are difficult to measure and subject to errors. With an attempt to standardize treatment response monitoring in cancer patients the European Organization for Research and Treatment of Cancer (EORTC) has developed quality of life questionnaires (EORTC QLQ-C30 version 3.0). They were validated in many clinical trials (Aaronson et al. 1993). They

Bone Seeking Radiopharmaceuticals for Metastatic Bone Pain 229

Last but not least the importance of imaging modalities should be mentioned with regard to individualized treatment monitoring. Functional rather than anatomical imaging techniques may be used to predict response. Several PET (Positron Emission Tomography) techniques are being developed for this purpose (John et al. 2008; Price et al. 2002). In fact, functional imaging will prove to be one of the major contributions of nuclear medicine to clinical oncology in the future. Does the treatment work? That question needs to be answered for each oncologic treatment on an individual basis. Nuclear imaging and PET in particular

Besides predictors of efficacy, predictors of toxicity are equally important for individualized patient management. Radiopharmaceuticals are important resources in the management of bone pain, but they need to be utilized in a manner that does not prevent other systemic therapy (Rago 1998). Thrombocytopenia is the dose limiting factor in treatment of painful bone metastases with bone seeking radiopharmaceuticals. De Klerk *et al* evaluated thrombocytopenia in patients with hormone refractory prostate carcinoma, treated with 186Re-HEDP (de Klerk et al. 1994). As an index of the extent of bone involvement, the bone scan index (BSI) was determined from the pre-treatment 99mTc-HDP scintigram. The BSI is a tool to describe the extent of skeletal metastases on a scale from 0 to 100% (Blake et al. 1986). They described a functional relation (r = 0.78; p < 0.001) of the percentage of platelet decrease after treatment with the extent and distribution of skeletal metastases (BSI) and administered activity, normalized to standard body surface area. Using this relation, it is possible to predict thrombocytopenia by pre-treatment skeletal scintigraphy and to adjust

the dosage for each patient to avoid unacceptable toxicity (de Klerk et al. 1994).

the influence of radiation on this process.

management.

However, more sophisticated indices of bone marrow function might also be of paramount importance. Recently, some very interesting reports have been published on 'reticulated platelets' (Briggs et al. 2004; Wang et al. 2002). In systemic radionuclide therapy, the megakaryocyte seems to be most vulnerable to radiation. It is of great interest to gain more knowledge of bone marrow function pre-treatment using 'biological' parameters like 'reticulated platelets'. These newly released platelets are larger and contain RNA. They were suggested to be the platelet analogue of the red cell reticulocyt. Assessment of platelet production using 'reticulated platelets' would distinguish between thrombocytopenia due to bone marrow failure and impending bone marrow recovery after cytotoxic therapy or thrombocytopenia due to increased peripheral platelet destruction and turnover. In both cases platelet levels are low, but in the latter 'reticulated platelet' levels will be high due to increased production (Wang et al. 2002). This noninvasive measurement could further increase our knowledge of platelet production and

By adding hematological, chemical and biological parameters, combined with the bone scan index, body surface area, administered activity and retained activity, an extended version of 'De Klerk's formula' may be developed. This is probably best done as a so called nomogram. Smaletz *et al* developed a nomogram to predict survival for patients with hormone refractory prostate carcinoma (Smaletz et al. 2002). A nomogram is a model in which individual parameters lead to a chance (from 0 to 100%) to experience a predefined outcome. The outcome may be defined as survival or for example toxicity. Such a model can be made to predict hematological toxicity (thrombocytopenia, leucopenia) after treatment with bone seeking radiopharmaceuticals to improve individualized patient

may be helpful.

include questions on global health status / quality of life, functional scales and symptom scales. The functional scales are subdivided in physical functioning, role functioning, emotional functioning, cognitive functioning and social functioning. The symptom scales are subdivided in fatigue, nausea and vomiting, pain, dyspnoea, insomnia, appetite loss, constipation, diarrhoea and financial difficulties. Each domain was scored on a scale of 0 – 100, according to the EORTC scoring manual (Fayers et al. 2001). The changes in scores may be used to monitor clinical effect.

Other often used clinical monitoring tools include the visual analogue scales (VAS) to monitor pain, changes in analgesic intake and, more basic, the physician's assessment based on anamnesis and physical examination. A matter of debate is the frequency of evaluation. It seems that daily assessment is necessary to appreciate the wide variation in clinical status that patients may experience from day to day (Han et al. 2002). In all cases it is difficult to find a good balance between accuracy and compliance. PSA is therefore still popular to measure an objective level of response (Bubley et al. 1999; Scher et al. 2008).

However, other predictors of response may prove to be much more reliable than PSA. In hormone-refractory prostate cancer patients in advanced stages of their disease the extent of metastatic disease in the skeleton is of high prognostic significance (Sabbatini et al. 1999). Furthermore it was shown that patients with advanced disease who had experienced a socalled Skeletal Related Event (defined as: pathologic fracture, spinal cord compression with vertebral compression fracture, the need for surgery to treat or prevent pathologic fractures or spinal cord compression, or the need for radiation to bone) had a significantly worse survival and poorer quality of life, in comparison with patients who had not experienced a Skeletal Related Event (DePuy et al. 2007). These results further confirm the importance to treat skeletal metastases adequately. Markers of skeletal metabolism were found to be related to outcome and survival. In several clinical studies in prostate cancer patients it was observed that markers of bone metabolism were able to predict outcome, both as absolute levels pretreatment and as changes after treatment (Jung et al. 2004; Lein et al. 2007). In large series of cancer patients, including a majority of prostate cancer patients, treated with zoledronic acid, it was found that baseline levels of the bone marker urinary N-terminal type I collagen peptide [NTX], as well as changes of NTX after treatment were able to predict improved survival (Coleman et al. 2005; Lipton et al. 2008). Other studies emphasized the importance of the bone marker serum bone specific alkaline phosphatase [BAP] as a predictor of outcome (Cook et al. 2006; Smith et al. 2007). Most of these studies used the same bone markers as we did in the study on the combined treatment of prostate cancer patients with 153Sm-EDTMP and zoledronic acid (i.e. NTX, BAP and serum procollagen type I N propeptide [PINP],). Although this study comprised a small patient population the results are of interest because they confirm the utility of these markers as predictors of clinical outcome, even in small numbers (Lam et al. 2007). Besides, they were first tested in the treatment monitoring of bone seeking radiopharmaceuticals. The bone formation markers BAP and PINP were in agreement with the clinical effect of the combined treatment regimen evaluated by EORTC questionnaires. The bone resorption marker NTX and PSA were not in agreement with the clinical effect. This supports the hypothesis that the extent of osteoblastic metastasis in hormone-refractory prostate cancer patients is an important parameter for clinical outcome. Both treatment itself and treatment monitoring should be directed to these osteoblastic metastases. Bone markers may well prove to be very useful predictors of clinical effect in the treatment with bone seeking radiopharmaceuticals. They should be used in future trials.

include questions on global health status / quality of life, functional scales and symptom scales. The functional scales are subdivided in physical functioning, role functioning, emotional functioning, cognitive functioning and social functioning. The symptom scales are subdivided in fatigue, nausea and vomiting, pain, dyspnoea, insomnia, appetite loss, constipation, diarrhoea and financial difficulties. Each domain was scored on a scale of 0 – 100, according to the EORTC scoring manual (Fayers et al. 2001). The changes in scores may

Other often used clinical monitoring tools include the visual analogue scales (VAS) to monitor pain, changes in analgesic intake and, more basic, the physician's assessment based on anamnesis and physical examination. A matter of debate is the frequency of evaluation. It seems that daily assessment is necessary to appreciate the wide variation in clinical status that patients may experience from day to day (Han et al. 2002). In all cases it is difficult to find a good balance between accuracy and compliance. PSA is therefore still popular to

However, other predictors of response may prove to be much more reliable than PSA. In hormone-refractory prostate cancer patients in advanced stages of their disease the extent of metastatic disease in the skeleton is of high prognostic significance (Sabbatini et al. 1999). Furthermore it was shown that patients with advanced disease who had experienced a socalled Skeletal Related Event (defined as: pathologic fracture, spinal cord compression with vertebral compression fracture, the need for surgery to treat or prevent pathologic fractures or spinal cord compression, or the need for radiation to bone) had a significantly worse survival and poorer quality of life, in comparison with patients who had not experienced a Skeletal Related Event (DePuy et al. 2007). These results further confirm the importance to treat skeletal metastases adequately. Markers of skeletal metabolism were found to be related to outcome and survival. In several clinical studies in prostate cancer patients it was observed that markers of bone metabolism were able to predict outcome, both as absolute levels pretreatment and as changes after treatment (Jung et al. 2004; Lein et al. 2007). In large series of cancer patients, including a majority of prostate cancer patients, treated with zoledronic acid, it was found that baseline levels of the bone marker urinary N-terminal type I collagen peptide [NTX], as well as changes of NTX after treatment were able to predict improved survival (Coleman et al. 2005; Lipton et al. 2008). Other studies emphasized the importance of the bone marker serum bone specific alkaline phosphatase [BAP] as a predictor of outcome (Cook et al. 2006; Smith et al. 2007). Most of these studies used the same bone markers as we did in the study on the combined treatment of prostate cancer patients with 153Sm-EDTMP and zoledronic acid (i.e. NTX, BAP and serum procollagen type I N propeptide [PINP],). Although this study comprised a small patient population the results are of interest because they confirm the utility of these markers as predictors of clinical outcome, even in small numbers (Lam et al. 2007). Besides, they were first tested in the treatment monitoring of bone seeking radiopharmaceuticals. The bone formation markers BAP and PINP were in agreement with the clinical effect of the combined treatment regimen evaluated by EORTC questionnaires. The bone resorption marker NTX and PSA were not in agreement with the clinical effect. This supports the hypothesis that the extent of osteoblastic metastasis in hormone-refractory prostate cancer patients is an important parameter for clinical outcome. Both treatment itself and treatment monitoring should be directed to these osteoblastic metastases. Bone markers may well prove to be very useful predictors of clinical effect in the treatment with bone

measure an objective level of response (Bubley et al. 1999; Scher et al. 2008).

seeking radiopharmaceuticals. They should be used in future trials.

be used to monitor clinical effect.

Last but not least the importance of imaging modalities should be mentioned with regard to individualized treatment monitoring. Functional rather than anatomical imaging techniques may be used to predict response. Several PET (Positron Emission Tomography) techniques are being developed for this purpose (John et al. 2008; Price et al. 2002). In fact, functional imaging will prove to be one of the major contributions of nuclear medicine to clinical oncology in the future. Does the treatment work? That question needs to be answered for each oncologic treatment on an individual basis. Nuclear imaging and PET in particular may be helpful.

Besides predictors of efficacy, predictors of toxicity are equally important for individualized patient management. Radiopharmaceuticals are important resources in the management of bone pain, but they need to be utilized in a manner that does not prevent other systemic therapy (Rago 1998). Thrombocytopenia is the dose limiting factor in treatment of painful bone metastases with bone seeking radiopharmaceuticals. De Klerk *et al* evaluated thrombocytopenia in patients with hormone refractory prostate carcinoma, treated with 186Re-HEDP (de Klerk et al. 1994). As an index of the extent of bone involvement, the bone scan index (BSI) was determined from the pre-treatment 99mTc-HDP scintigram. The BSI is a tool to describe the extent of skeletal metastases on a scale from 0 to 100% (Blake et al. 1986). They described a functional relation (r = 0.78; p < 0.001) of the percentage of platelet decrease after treatment with the extent and distribution of skeletal metastases (BSI) and administered activity, normalized to standard body surface area. Using this relation, it is possible to predict thrombocytopenia by pre-treatment skeletal scintigraphy and to adjust the dosage for each patient to avoid unacceptable toxicity (de Klerk et al. 1994).

However, more sophisticated indices of bone marrow function might also be of paramount importance. Recently, some very interesting reports have been published on 'reticulated platelets' (Briggs et al. 2004; Wang et al. 2002). In systemic radionuclide therapy, the megakaryocyte seems to be most vulnerable to radiation. It is of great interest to gain more knowledge of bone marrow function pre-treatment using 'biological' parameters like 'reticulated platelets'. These newly released platelets are larger and contain RNA. They were suggested to be the platelet analogue of the red cell reticulocyt. Assessment of platelet production using 'reticulated platelets' would distinguish between thrombocytopenia due to bone marrow failure and impending bone marrow recovery after cytotoxic therapy or thrombocytopenia due to increased peripheral platelet destruction and turnover. In both cases platelet levels are low, but in the latter 'reticulated platelet' levels will be high due to increased production (Wang et al. 2002). This noninvasive measurement could further increase our knowledge of platelet production and the influence of radiation on this process.

By adding hematological, chemical and biological parameters, combined with the bone scan index, body surface area, administered activity and retained activity, an extended version of 'De Klerk's formula' may be developed. This is probably best done as a so called nomogram. Smaletz *et al* developed a nomogram to predict survival for patients with hormone refractory prostate carcinoma (Smaletz et al. 2002). A nomogram is a model in which individual parameters lead to a chance (from 0 to 100%) to experience a predefined outcome. The outcome may be defined as survival or for example toxicity. Such a model can be made to predict hematological toxicity (thrombocytopenia, leucopenia) after treatment with bone seeking radiopharmaceuticals to improve individualized patient management.

Bone Seeking Radiopharmaceuticals for Metastatic Bone Pain 231

Aaronson NK et al (1993) The European Organization for Research and Treatment of Cancer

Attard G et al (2008) Phase I clinical trial of a selective inhibitor of CYP17, abiraterone

Beer TM et al (2007) Double-blinded randomized study of high-dose calcitriol plus docetaxel

Berlin NI (2000) Treatment of the myeloproliferative disorders with 32P. *Eur.J.Haematol.* 65

Berthold DR et al (2008) Docetaxel plus prednisone or mitoxantrone plus prednisone for

Berthold DR, Sternberg CN, and Tannock IF (2005) Management of advanced prostate

Blake GM et al (1986) Sr-89 therapy: strontium kinetics in disseminated carcinoma of the

Boyle BJ et al (2001) Insulin-like growth factor binding protein-3 mediates 1 alpha,25-

Briggs C et al (2004) Assessment of an immature platelet fraction (IPF) in peripheral

Bubley GJ et al (1999) Eligibility and response guidelines for phase II clinical trials in

Buchan RC and Brindle JM (1970) Radioiodine therapy to out-patients - the contamination

Carducci MA et al (2003) Effect of endothelin-A receptor blockade with atrasentan on tumor

Chapman CR and Gavrin J (1999) Suffering: the contributions of persistent pain. *Lancet* 353

Cheung A and Driedger AA (1980) Evaluation of radioactive phosphorus in the palliation of

Coebergh, J. W. W., van de Poll-Franse, L. V., and Alers, J. C. Kanker in Nederland, Trends, prognoses en implicaties voor zorgvraag. Amsterdam. KWF , 65-73. 2004.

Specific Antigen Working Group. *J.Clin.Oncol.* 17 (11):3461-3467.

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Auclerc G et al (2000) Management of advanced prostate cancer. *Oncologist.* 5 (1):36-44. Bauer KS et al (1999) A pharmacokinetically guided Phase II study of carboxyamido-triazole in androgen-independent prostate cancer. *Clin.Cancer Res.* 5 (9):2324-2329. Bauman G et al (2005) Radiopharmaceuticals for the palliation of painful bone metastasis-a

report from the ASCENT Investigators. *J.Clin.Oncol.* 25 (6):669-674.

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through p21/WAF1. *J.Urol.* 165 (4):1319-1324.

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dihydroxyvitamin d(3) growth inhibition in the LNCaP prostate cancer cell line

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(2):242-245.


*3.2. The Role of the Spanish Insurance Company, Insurance Compensation Consortium (Consorcio de Compensación de Seguros, CCS)*

The high loss potential from natural hazards and the need to make more generalized insurance cover viable has led many countries to involve the state in specific cover schemes, collaborating to varying degrees with the private market. The CCS is a government institution attached to the current Ministry of Economy Affairs and Digital Transformation. This institution has its legal personality and full capacity to act, and is subject to private law. This means that, although it is a government institution, it is subject to the rules contained in the legislation establishing the legal regulation and supervision of private insurance.

(**b**)

**Figure 3.** CCS's payouts due to damages caused by pluvial floods in Barcelona: (**a**) historical total annual amounts (1996 to 2018); (**b**) total historic (22 years) amounts grouped into diverse types of properties, according to CCS classification.

**Figure 4.** Consequences of pluvial floods that occurred in Barcelona on (**a**) 9 October 2018, and (**b**) 15 November 2018. Sources: a) https://www.elperiodico.com, and b) https://www.telecinco.es.

(**a**) (**b**)

The CCS covers so-called extraordinary risks, which include both natural hazards and those of a political or social nature, not expressly assumed by the insurance company issuing the standard policy. Its coverage encompasses losses derived from direct damage caused to people or to property, as well as business interruption due to the alteration of normal outcomes of production or business processes concerning such activity. The coverage of extraordinary risks is compulsory and is necessarily linked to underwriting (an insurance policy) in certain branches of insurance related to property (vehicles, home, etc.) and persons (life, accidents, etc.). All insurance policies rates include a surcharge to the

endowment of a common fund, under the principle of solidarity. Claims must be lodged within seven days following the damage event to the private insurer when it expressly covers the extraordinary event that caused the damage. Otherwise, the CCS will be responsible for the payouts, and could receive the claim directly from the policyholder or through the private insurer. A policyholder will be entitled to compensation after the damage assessment of an expert surveyor designated by the CCS.

When an extraordinary risk occurs, such as the pluvial flood events that hit Barcelona on 9 October and 15 November 2018 (Figure 4), the CCS sends one or more expert surveyors to provide an estimation of the extent of the damage. According to discussions with the CCS, these estimations are extremely close to the final payouts, which denotes the remarkable know-how of such experts. For these reasons, the depth-damage curves developed in this study are mainly based on the knowledge of a flood expert surveyor. In the same line, the curves developed by USACE [51] were based on the expert opinion method described in the Handbook of Forecasting Techniques (IWR Contract Report 75-7, December 1975) [52] and the Handbook of Forecasting Techniques, Part II, Description of 31 Techniques (Supplement to IWR Contract Report 75-7, August 1977) [53].


*4.2. Experiment B*

To fully demonstrate the performance of BDS-3 PPP-B2b/INS loosely coupled integration in an urban scenario, we conducted Experiment B in a complex urban environment. Experiment B was carried out from 09:00:00 to 09:49:59 on 5 December 2021 in GPS time. The trajectory of the land vehicle is shown in Figure 7.

**Figure 7.** The trajectory of the land vehicle in Experiment B.

Figure 8 presents the number of visible BDS-3/GPS satellites and the values of PDOP. The number of available BDS-3/GPS satellites dropped frequently, and at the same time the PDOP value significantly increased as several typical city features such as tall buildings, trees, and over-bridges appeared along the vehicle route.

During the first 35 min, there were enough satellites for the implementation of PPP-B2b positioning. The positioning errors of PPP-B2b only, PPP-B2b/MEMS-IMU loosely coupled integration and PPP-B2b/tactical-IMU loosely coupled integration are plotted in Figure 9. It can be found that all three schemes provided similar positioning accuracy in this 35-min period. The RMS values of positioning errors were 20.7 cm, 17.3 cm, and 47.6 cm in the east, north, and up directions for PPP-B2b only. For PPP-B2b/INS loosely coupled integration, the corresponding RMS values were 20.8 cm, 17.4 cm, and 47.5 cm for the PPP-B2b/MEMS-IMU scheme, and 20.3 cm, 17.4 cm, and 47.5 cm for the PPP-B2b/tactical-IMU scheme. It should be noted that a convergence period of 15 min was removed when we

computed the positioning accuracy. Clearly, the equipped IMU sensors could not improve the performance of the PPP-B2b service when there was no GNSS signal blockage.

**Figure 8.** Number of visible BDS-3/GPS satellites and PDOPs in Experiment B.

**Figure 9.** Positioning errors of PPP-B2b only (**top** panel), PPP-B2b/MEMS-IMU loosely coupled integration (**middle** panel), and PPP-B2b/tactical−IMU loosely coupled integration (**bottom** panel) during the period from 09:00:00 to 09:34:59.

From 09:35:00 to the end of Experiment B, the land vehicle crossed two over-bridges, as shown in Figure 10. Two GNSS signal outages appeared when the land vehicle crossed the bridges, lasting 15 s and 5 s, respectively. During these two GNSS signal outages, PPP-B2b positioning could be implemented, because the number of visible satellites was less than four. Therefore, the PPP-B2b positioning was re-converged after crossing the bridges. Figure 11 shows the positioning errors of PPP-B2b only, PPP-B2b/MEMS-IMU loosely coupled integration, and PPP-B2b/tactical-IMU loosely coupled integration. It can be seen that the positioning accuracy of PPP-B2b/INS loosely coupled integration exhibited better performance than PPP-B2b positioning during the re-convergence phase. The overall positioning accuracies for the three schemes were calculated from 09:35:00 to 09:49:59 and are presented in Table 5. The RMS values of positioning errors for the east, north, and up components were 114.2 cm, 200.0 cm, and 244.3 cm for PPP-B2b only. These figures improved to 41.0 cm, 44.5 cm, and 64.0 cm for PPP-B2b/MEMS-IMU loosely coupled integration, and to 41.2 cm, 41.0 cm, and 62.5 cm for PPP-B2b/tactical-IMU loosely coupled integration. Obviously, the performance of PPP-B2b/INS loosely coupled integration was significantly improved compared to PPP-B2b only in the last 15-min period of Experiment B. The positioning accuracy of PPP-B2b/tactical-IMU loosely coupled integration was slightly better than that of PPP-B2b/MEMS-IMU loosely coupled integration.

**Figure 10.** Two bridge scenarios during the period from 09:35:00 to 09:49:59.

**Table 5.** The positioning accuracy during the period from 09:35:00 to 09:49:59 (unit: cm).

**Figure 11.** Positioning errors of PPP-B2b only (**top** panel), PPP-B2b/MEMS-IMU loosely coupled integration (**middle** panel), and PPP-B2b/tactical−IMU loosely coupled integration (**bottom** panel) during the period from 09:35:00 to 09:49:59.

#### **5. Conclusions**

Since July 2020, PPP-B2b has served as a featured service of BDS-3 to support real-time PPP. Compared to the IGS RTS service, PPP-B2b is a satellite-based service, which is not limited by Internet communication. Its high-precision positioning performance shows great potential in real-time GNSS applications. However, the BDS-3 PPP-B2b service continues to encounter challenges in urban environments due to GNSS signal outages. In order to overcome the drawbacks of the BDS-3 PPP-B2b service in urban environments, we set up a BDS-3 PPP-B2b/INS loosely coupled integration system and evaluated its performance in different urban scenarios.

The experimental results indicate that PPP-B2b/INS loosely coupled integration cannot improve the positioning performance in open-sky environments where there are enough GPS/BDS-3 satellites for PPP-B2b positioning. However, PPP-B2b/INS loosely coupled integration can show its superiority when GNSS signal outage occurs. During GNSS signal outages, the INS mechanization can provide continuous positioning. Therefore, the performance of PPP-B2b positioning can be improved by adopting PPP-B2b/INS loosely coupled integration in GNSS blockage environments. In the first experiment, we simulated four 30-s periods of GNSS signal outages. At the last epoch of the simulated outages, the averaged

positioning errors in the east, north, and up directions were 300.0 cm, 498.0 cm, and 41.0 cm, respectively, for PPP-B2b/MEMS-IMU loosely coupled integration, and 18.6 cm, 21.8 cm, and 6.1 cm, respectively, for PPP-B2b/tactical-IMU loosely coupled integration. In addition, we also evaluated the performance of PPP-B2b/INS loosely coupled integration in a real complex urban environment. When the land vehicle crossed bridges, two GNSS signal interruptions appeared that lasted 15 s and 5 s, respectively. Compared to PPP-B2b only, the respective improvement of positioning accuracy in the east, north, and up components was 64.1%, 77.8%, and 73.8% for PPP-B2b/MEMS-IMU loosely coupled integration, and 63.9%, 79.5%, and 74.4% for PPP-B2b/Tactical-IMU loosely coupled integration.

It can be concluded that when there are sufficient valid satellites for PPP-B2b positioning, the positioning accuracy of PPP-B2b/INS loosely coupled integration depends mainly on the accuracy of PPP-B2b. In urban environments, positioning performance can be significantly improved compared to PPP–B2b alone, by adopting PPP-B2b/INS loosely coupled integration especially in GNSS signal blockage environments.

**Author Contributions:** X.X.; Conceptualization, methodology, software, visualization, and writing original draft preparation. Z.N.; Conceptualization, software, writing—review and editing, and funding acquisition. Z.W.; writing—review and editing, supervision, project administration, and funding acquisition. B.W.; validation and formal analysis. Q.D.; validation and formal analysis. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was supported by the National Natural Science Foundation of China (No. 42104011), the Shandong Provincial Natural Science Foundation (No. ZR2021QD069), the National Key Research and Development Program of China (No. 2019YFC1509205), and Sinopec Science and Technology Entry Program (JP210004).

**Data Availability Statement:** The datasets used in this study are managed by the College of Oceanography and Space Informatics, China University of Petroleum, Qingdao, China and can be available on request from the corresponding author.

**Conflicts of Interest:** The authors declare no conflict of interest.