**2. Bisphosphonates**

#### **2.1. Definition**

Bisphosphonates are non-metabolizable pyrophosphate analogues that are deposited on the bones and prevent or improve bone complications of the patients with bone alterations. Different bisphosphonates differ depending on the alterations of the R-2 lateral chain structure. These R-2 lateral chains determine the efficiency and the cellular effects of the inhibition of bone resorption [73]. The bisphosphonates are internalized by osteoclasts, causing the interruption of bone resorption. They also have antigiogenic properties, since they reduce the circulating levels of vascular endothelial growth factor (VEGF) [74–78] and antineoplastic effects [79]. It has very little intestinal absorption and is excreted without being metabolized by the kidneys.

Until 2001, pamidronate (Aredia®) was the only drug approved in the USA for the treatment of bone metastasis. In 2002, the US Food and Drug Administration (FDA) [80] approved zoledronic acid (Zometa®) as a treatment for these patients. Currently, the annual transfusion of zoledronate (Reclast®) and the parenteral formulation of ibandronate (Bonviva®) administered every 3 months has been approved by the FDA for the treatment of osteoporosis [81].

In 2003, using the articles by Marx [12] and Ruggiero et al. [25], they observed and reported on the cases of unhealed bone exposure in the maxillofacial region in patients treated with IV bisphosphonates and, after multiple further publications, in September 2004, Novartis the manufacturer of pamidronate (Aredia®) and zoledronic acid (Zometa), added the complications deriving from this treatment to the labeling of these drugs, with the purpose of warning the health care professionals of the possibility of developing osteonecrosis of the jaws [82].

#### **2.2. Types of bisphosphonates**

According to their chemical structure, these are divided into two groups.

**Non-nitrogenous:** These are very similar to natural pyrophosphate, such as etidronate or clodronate that contain CH3 and Cl groups instead of the R2 chain and a nitrogen-free ring, such as tiludronate. They are metabolized by macrophages in toxic analogues of adenosine triphosphate (ATP) [83].

**Nitrogenous:** They present higher power, such as zoledronate and pamidronate, with a primary atom of basic nitrogen with an alkyl chain. They have a power 10–100 times higher than non-nitrogenous. Mevalonate inhibits the cholesterol route through the farnesyl diphosphate synthase enzyme [83].

There is a great variety of bisphosphonates approved for clinical use in the USA (**Table 4**).

Osteonecrosis of the Jaws. Prevalence, Risk Factors and Role of Microbiota and Inflammation... http://dx.doi.org/10.5772/intechopen.69315 61


**Table 4.** Drugs approved in EEUU.

#### *2.2.1. IV bisphosphonates*

debridement. The decision to suppress the drug must be assessed by the physician and the

Bisphosphonates are non-metabolizable pyrophosphate analogues that are deposited on the bones and prevent or improve bone complications of the patients with bone alterations. Different bisphosphonates differ depending on the alterations of the R-2 lateral chain structure. These R-2 lateral chains determine the efficiency and the cellular effects of the inhibition of bone resorption [73]. The bisphosphonates are internalized by osteoclasts, causing the interruption of bone resorption. They also have antigiogenic properties, since they reduce the circulating levels of vascular endothelial growth factor (VEGF) [74–78] and antineoplastic effects [79]. It has very little intestinal absorption and is excreted without being metabolized

Until 2001, pamidronate (Aredia®) was the only drug approved in the USA for the treatment of bone metastasis. In 2002, the US Food and Drug Administration (FDA) [80] approved zoledronic acid (Zometa®) as a treatment for these patients. Currently, the annual transfusion of zoledronate (Reclast®) and the parenteral formulation of ibandronate (Bonviva®) administered every 3 months has been approved by the FDA for the treatment of osteoporosis [81].

In 2003, using the articles by Marx [12] and Ruggiero et al. [25], they observed and reported on the cases of unhealed bone exposure in the maxillofacial region in patients treated with IV bisphosphonates and, after multiple further publications, in September 2004, Novartis the manufacturer of pamidronate (Aredia®) and zoledronic acid (Zometa), added the complications deriving from this treatment to the labeling of these drugs, with the purpose of warning the health care professionals of the possibility of developing osteonecrosis of the jaws [82].

**Non-nitrogenous:** These are very similar to natural pyrophosphate, such as etidronate or clodronate that contain CH3 and Cl groups instead of the R2 chain and a nitrogen-free ring, such as tiludronate. They are metabolized by macrophages in toxic analogues of adenosine

**Nitrogenous:** They present higher power, such as zoledronate and pamidronate, with a primary atom of basic nitrogen with an alkyl chain. They have a power 10–100 times higher than non-nitrogenous. Mevalonate inhibits the cholesterol route through the farnesyl diphosphate

There is a great variety of bisphosphonates approved for clinical use in the USA (**Table 4**).

According to their chemical structure, these are divided into two groups.

patient, as long as the systemic conditions allow.

**2. Bisphosphonates**

**2.1. Definition**

60 Osteonecrosis

by the kidneys.

**2.2. Types of bisphosphonates**

triphosphate (ATP) [83].

synthase enzyme [83].

It is estimated that over 2.8 million cancer patients from around the world have received treatment with IV bisphosphonates since their introduction [84].


It is a heterocyclic imidazole and, to date, the most powerful bisphosphonate to be administered to humans. In a test on in-vitro bone resorption using mouse cranium, zoledronate was at least 100 times more powerful than pamidronate. Additionally, the in-vivo animal model of calcitriol-induced hypercalcemia in thyroparathyroidectomized rats was 850 times more active than pamidronate and over 4 times more powerful than clodronate. Of all the bisphosphonates under clinical evaluation, this is the one having a higher therapeutic relationship between the desired inhibition of bone resorption and the undesired inhibition of bone mineralization, furthermore, several toxicology studies proved that the compound is safe [85].

#### *2.2.2. Oral bisphosphonates*

Due to the proven clinical effectiveness, it is considered a first line therapy in the treatment of osteoporosis and they are the most prescribed antiresorptive agents.

• **Alendronate** (Fosamax): 70 mg once a week during the osteoporosis treatment or less, if it has been prescribed for the prevention of osteoporosis.

It has been proven to prevent approximately 50% of cases in the prevention of the bone loss in the spine or hips in menopausal women and the reduction of bone fractures [86, 87].

• **Risedronate** (Actonel): 35 mg once a week.

In a prospective study with a large sample, risedronate produced a reduction of 30% in hip fractures [88, 89].


#### **2.3. Metabolism**

Bisphosphonates have an average life between 30 min and 2 h and are deposited in the locations with higher bone metabolism, and can remain at the bone level for approximately 10 years. The highest concentration is located in urine and saliva, and the most frequent adverse effects are renal insufficiency and osteonecrosis [90–92]. The accumulative doses described by Maerevoet, is of 72 mg for 18 months [90, 93].

In the case of IV bisphosphonates, such as zolendronic acid, 40% is eliminated unaltered through urine after 24 h [94] and the remaining 60% are united at bone level due to the great affinity of hydroxyapatite. This phenomenon takes place in areas of bone remodeling, in which the periodical exchange produces unaltered kidney excretion after a long elimination phase [95]. The mean life of this second elimination phase may last months or years, depending on the duration of the bisphosphonate treatment [96].

Excretion of oral bisphosphonates has also been studied, for example, in a rat study, in which they administered risedronate orally, and determined that 80% of the drug was excreted through the kidneys 12 h after the administration of the drug. Additionally, the study concluded that taking oral bisphosphonates with mineral water that contains high calcium and magnesium levels reduced the effect of the drug; therefore it is advisable to take it as soon as the patient wakes up and in a vertical position [97]. These authors previously described the effect of water in combination with taking alendronate [98].

#### **2.4. Detection of bisphosphonates**

The concentration of bisphosphonates in a specific bone location depends on the speed of bone remodeling and blood circulation [99]. It is important to know the concentration of accumulated bisphosphonate in the bone to understand the long-term drug effect and its toxicity. Today, numerous authors have been able to quantify these drugs in plasma and urine through mass spectrometry (MS), which requires a previous derivation process that allows to transform the bisphosphonates into more hydrophobic substances so they can be studied [100, 101].

The development of analysis methods for the detection of bisphosphonates in biological matrices, it is hard due to the chemical properties of these compounds. The detection of bisphosphonates in human biological matrices comes with certain difficulties and therefore a broad range of analytic techniques have been described, such as gas chromatography [102], ion chromatography [103], capillary electrophoresis, ionization mass spectrometry by electrospray [104] and chromatography of fluids [105].

#### *2.4.1. Mass spectrometry (MS)*

In a prospective study with a large sample, risedronate produced a reduction of 30% in hip

• **Ibandronate** (Boniva): is the latest drug to be approved by the FDA (March, 2005) for the

• **Etidronate** (Didronel): prescribed for the Paget disease with a dose of 300–750 mg/day for

• **Tiludronate** (Skelid): prescribed for Paget disease with a dose of 400 mg/day for 3 months.

Bisphosphonates have an average life between 30 min and 2 h and are deposited in the locations with higher bone metabolism, and can remain at the bone level for approximately 10 years. The highest concentration is located in urine and saliva, and the most frequent adverse effects are renal insufficiency and osteonecrosis [90–92]. The accumulative doses described by

In the case of IV bisphosphonates, such as zolendronic acid, 40% is eliminated unaltered through urine after 24 h [94] and the remaining 60% are united at bone level due to the great affinity of hydroxyapatite. This phenomenon takes place in areas of bone remodeling, in which the periodical exchange produces unaltered kidney excretion after a long elimination phase [95]. The mean life of this second elimination phase may last months or years, depend-

Excretion of oral bisphosphonates has also been studied, for example, in a rat study, in which they administered risedronate orally, and determined that 80% of the drug was excreted through the kidneys 12 h after the administration of the drug. Additionally, the study concluded that taking oral bisphosphonates with mineral water that contains high calcium and magnesium levels reduced the effect of the drug; therefore it is advisable to take it as soon as the patient wakes up and in a vertical position [97]. These authors previously described the

The concentration of bisphosphonates in a specific bone location depends on the speed of bone remodeling and blood circulation [99]. It is important to know the concentration of accumulated bisphosphonate in the bone to understand the long-term drug effect and its toxicity. Today, numerous authors have been able to quantify these drugs in plasma and urine through mass spectrometry (MS), which requires a previous derivation process that allows to transform the bisphosphonates into more hydrophobic substances so they can be studied

The development of analysis methods for the detection of bisphosphonates in biological matrices, it is hard due to the chemical properties of these compounds. The detection of bisphosphonates in human biological matrices comes with certain difficulties and therefore a

treatment of osteoporosis and it is administered monthly.

Maerevoet, is of 72 mg for 18 months [90, 93].

ing on the duration of the bisphosphonate treatment [96].

effect of water in combination with taking alendronate [98].

**2.4. Detection of bisphosphonates**

[100, 101].

fractures [88, 89].

62 Osteonecrosis

6 months.

**2.3. Metabolism**

A method for the extraction and detection of zoledronic acid in urine and blood plasma or even accumulated in the bone (in a mouse model) through the combination of chromatography and mass spectrometry (MS) [100, 106].

In these studies, a higher accumulation of bisphosphonates in bone extracts of the mandible was detected, compared to other types of bones [106]. On the other hand, human urine and blood plasma detected a maximum concentration peak of the drug of 77 μM (5 h after the administration) and 1.5 μM (after 1 h), respectively [100]. This methodology achieves high sensitivity and specificity in the detection, however, it requires a pretty complex and arduous treatment of the sample requiring phases with chemical reactions to derive the complex. The complexity of the treatment of the sample can be a limiting factor when the number of samples to be analyzed is high, as in the case of the follow-up of the pharmacokinetics and the bioavailability of zoledronic acid since its administration. For this reason, it would be desirable to have a more efficient alternative method to detect this drug.

#### *2.4.2. Nuclear magnetic resonance spectroscopy (NMR)*

Nuclear magnetic resonance (NMR) spectroscopy is a useful technique in chemometrics that can be used for the characterization of simple or complex mixes of different sources and provide quantitative results. There is a great variety of "omics" applications for NMR, such as *metabonomics, metabolomics, proteomics, transcriptomics, fluxomics, foodomics, lipidomics*, *fermentanomics*, *isotopomics*, etc. One of the areas with greater impact in biomedicine is metabolomics by NMR for the study of metabolites in different types of samples, such as biofluids (urine, saliva, blood plasma, blood serum, sweat, etc.), tissue extracts, cerebrospinal fluid, cells, etc. Metabolomics by NMR can be used to find biomarkers for the study, diagnosis and prognosis of diseases. Similarly, it can help many traditional analysis at a clinical level, since the cost per sample is or can be competitive if large lots of samples are managed, in terms of the time for the analysis (measurement) and the generation of results (automated). There are a number of pathologies for which the study of biofluids or tissues by NMR has found useful biomarkers [107, 108]. A relevant case of application of NMR in this area is the quantification of lipoprotein in blood serum/plasma. NMR offers details on the relative abundance of different subclasses of lipoproteins that are not accessible to traditional analysis methods [109, 110].

It is a quantitative technique that allows determination of the absolute concentration of diluted substances in general, including biofluids. It is based on the fact that the intensity of a signal in the NMR spectrum is proportionate to the concentration of the molecule (or metabolite) generating the said signal. As an approximate value, to assess the sensitivity of this technique, in a current spectrometer with a 11.7 T magnet, the minimum concentration required to detect a molecule in a monodimensional spectrum (1D) of 1 H (proton spectrum, as it is commonly known in our jargon) must amount to a minimum of 10 μm, for a measurement period of 15 min.

For the case of urine samples, under these same measurement conditions, 50–60 metabolites can be identified and quantified in their proton spectrum (**Figure 2**).

**Figure 2.** Typical spectrum of 1 H NMR of a human urine sample. The numbers indicate the following metabolites: 1: creatinine, 2: citric acid, 3: glycine, 4: formic acid, 5: methanol, 6: guanidinoacetic acid, 7: acetic acid, 8: l-cysteine, 9: glycolic acid, 10: creatine, 11: Isocitric acid, 12: hippuric acid, 13: l-glutamine, 14: l-alanine, 15: l-Lysine, 16: gluconic acid, 17: 2-hidoxiglutaric acid, 18: d-glucose, 19: indoxyl sulfate, 20: trimethyl-N-oxide, 21: ethanolamine, 22: l-lactic acid, 23: taurine, 24: l-threonine, 25: dimethylamine, 26: pyroglutamic acid, 27: trigonelline, 28: sucrose, 29: trimethylamine, 30: mannitol, 31: l-serine, 32: acetone, 33: l-cystine, 34: adipic acid, 35: l-histidine, 36: l-tyrosine, 37: imidazole, 38: mandelic acid, 39: dimethylglycine, 40: cis-aconitic acid, 41: urea, 42: 3-(3-Hydroxyphenyl)-3-hydroxypropanoic (HPHPA), 43: phenol, 45: isobutyric acid, 46: methylsuccinic acid, 47: 3-Aminoisobutíric acid, 48: l-fucose, 49: N-acetylaspartic acid, 50: N-acetylneuraminic, 51: acetoacetic acid, 52: Alpha-aminoadipic acid, 53: methylguanidine, 54: phenylacetylglutamine [111].

When a series of NMR spectra need to be compared, such as urine, it is important to consider some details regarding post-processing of the spectra and the process to obtain their quantitative values, in such a way that no errors are introduced in this phase and the quantitative results that are obtained are the most precise and repeatable as possible. Although with slight variations regarding some details, the general method for the treatment of spectral information for the metabolomic studies by NMR is the one described below.

#### *2.4.2.1. Preparation of a biofluid sample*

as it is commonly known in our jargon) must amount to a minimum of 10 μm, for a measure-

For the case of urine samples, under these same measurement conditions, 50–60 metabolites

H NMR of a human urine sample. The numbers indicate the following metabolites:

1: creatinine, 2: citric acid, 3: glycine, 4: formic acid, 5: methanol, 6: guanidinoacetic acid, 7: acetic acid, 8: l-cysteine, 9: glycolic acid, 10: creatine, 11: Isocitric acid, 12: hippuric acid, 13: l-glutamine, 14: l-alanine, 15: l-Lysine, 16: gluconic acid, 17: 2-hidoxiglutaric acid, 18: d-glucose, 19: indoxyl sulfate, 20: trimethyl-N-oxide, 21: ethanolamine, 22: l-lactic acid, 23: taurine, 24: l-threonine, 25: dimethylamine, 26: pyroglutamic acid, 27: trigonelline, 28: sucrose, 29: trimethylamine, 30: mannitol, 31: l-serine, 32: acetone, 33: l-cystine, 34: adipic acid, 35: l-histidine, 36: l-tyrosine, 37: imidazole, 38: mandelic acid, 39: dimethylglycine, 40: cis-aconitic acid, 41: urea, 42: 3-(3-Hydroxyphenyl)-3-hydroxypropanoic (HPHPA), 43: phenol, 45: isobutyric acid, 46: methylsuccinic acid, 47: 3-Aminoisobutíric acid, 48: l-fucose, 49: N-acetylaspartic acid, 50: N-acetylneuraminic, 51: acetoacetic acid, 52: Alpha-aminoadipic acid, 53: methylguanidine, 54: phenylacetylglutamine [111].

can be identified and quantified in their proton spectrum (**Figure 2**).

ment period of 15 min.

64 Osteonecrosis

**Figure 2.** Typical spectrum of 1

A series of protocols have been established for the preparation of biofluid samples and their subsequent preservation until the NMR measuring [112, 113]. Similarly, there are experimental parameters to be used for the measurement of the NMR spectrum for each type of biofluid [112, 113]. These protocols standardize the measurements, thus allowing the comparison of the spectra obtained and the NMR spectrum (e.g. the Human Metabolome Database: www. hmdb.ca) and/or with bibliographic results. These sample preparation protocols tend to be simple and do not require special laboratory equipment.

#### *2.4.2.1.1. Generation of a signal intensity data matrix (integrals) on regions of interest (ROIs) of the NMR spectrum*

The raw data provided by the spectrometer when measuring the 1 H NMR spectrum is called free induction decay (FID). The data must be post-processed by applying a series of operations to generate a final spectrum with a scale of frequencies (expressed by standardized ppm units) and with the best quality possible [114]. Once the post-processing has been completed for each of the spectra to be studied, the following phase consists of comparing analogue regions of the spectrum with the target to find, if possible, a certain region that could serve as a biomarker, in other words, a region in which the signal's intensity pattern is significantly different in the samples of the control group and the experiment group, while being similar within their own group.

Instead of manually selecting one or several regions for comparison, the usual and practical procedure is to perform a systematic analysis, dividing the entire spectrum automatically, from right to left, through a series of small segmental regions (e.g. with an established width) in which the area of the signal is integrated individually. Each of these regions is called buckets or regions of interest (ROIs). This way, a complete spectrum is represented as a data vector formed by the integrals of the selected ROIs, with as many integral values as segments of the spectrum have been created.

Lastly, a table is created with the data, placing the vectors of each of the analyzed samples in different lines (**Figure 3**). There are software tools that automate these operations to generate the data table, as well as the phase before the post-processing.

#### *2.4.2.2. Standardization of the data of the ROIs Matrix*

Of the ROI matrix integral values, the negative values are initially purged since they only contain noise (a ROI must have at least an integral value or a signal area that equals zero).

**Figure 3.** Scheme for the construction of a NMR ROI data matrix from 53 NMR spectra. Above: 53 1 H spectra in which we have selected certain ROIs for the integration of the signal. The vertical piling scale identifies each of the samples (# sample). The same color has been used to identify samples that should be, a priori, of the same type. Below: NMR ROIs data matrix obtained through the integration of each of the ROIs.

The ROIs data standardization process seeks to take all the analyzed samples to a "virtual" constant that eliminates all the differences in global concentration in the ROI integral samples, which could simply respond to the urine having a higher or lower concentration of H2 O. When the dilution effect is eliminated, the differences between values of the same ROI in a pair of samples directly reflect variations in the relative concentration of the studied metabolite.

#### *2.4.2.2.1. Standardization method of each ROI line at a standard addition*

A first standardization method consists of standardizing the total addition of the ROIs. This method considers that the total addition of all the ROIs of the same spectrum has a constant value, and that this is the same for all the samples. This type of standardization by matrix lines consists of dividing each ROI value by the total addition of ROIs of its same line. As a result, the standardized ROIs of a sample add up to the unit.

#### *2.4.2.2.2. Probabilistic quotient standardization method*

This method consists of using the data of the ROIs of a certain sample of the study (a line of the matrix) and dividing the ROIs between those of another sample that is used as reference. This allows us to deduce the most probable multiplication factor so that the ROIs of the first sample are as close as possible to the reference sample. The most probable multiplication factor is calculated for each sample (for each line of the matrix). As a result of applying each factor on the ROIs of the relevant samples, their ROIs will be standardized as if the sample would have been prepared in the same concentration as the reference sample [115].

#### *2.4.2.3. Statistical analysis of the NMR ROIs Matrix*

The Matrix of standardized ROIs is analyzed by multivariate statistic methods to identify the ROIs that contain similar or different patterns of the integral in samples of the same or different group and their potential biomarkers. To do this, algorithms, such as the Principal Components Analysis (PCA), Discriminant Analysis (DA) and Orthogonal Projections to Latent Structures-Discriminant Analysis (OPLS-DA) are used. These algorithms have been implemented in several general statistical software packages, for example, R, SPSS or XLSLAT, among others.

In favorable cases, one or more ROIs with potential biomarkers related to the property under investigation are identified, for example, the effect of certain medical treatments, a type of diet, a disease, etc. Additional objectives can then be included in the study, such as the identification of what specific biomarkers (metabolites) were altered compared to the control group, to try to discover through which metabolic pathway they underwent this change. Some of the biomarkers that are identified as different from the control group may not correspond to any of those that are typically found in the relevant type of biofluid, but they may proceed from the metabolism of a drug or a special diet that has been prescribed. In this case, once its NMR signal has been identified, we could consider performing a longitudinal follow-up on its appearance in the biofluid since its administration.

#### **2.5. Medication-related osteonecrosis of the jaw (MRONJ)**

Currently, two types of drugs, aside from bisphosphonates, have been proven to cause necrosis of the jaws.

#### *2.5.1. Antireabsorptives: denosumab*

The ROIs data standardization process seeks to take all the analyzed samples to a "virtual" constant that eliminates all the differences in global concentration in the ROI integral samples,

we have selected certain ROIs for the integration of the signal. The vertical piling scale identifies each of the samples (# sample). The same color has been used to identify samples that should be, a priori, of the same type. Below: NMR ROIs

the dilution effect is eliminated, the differences between values of the same ROI in a pair of samples directly reflect variations in the relative concentration of the studied metabolite.

A first standardization method consists of standardizing the total addition of the ROIs. This method considers that the total addition of all the ROIs of the same spectrum has a constant value, and that this is the same for all the samples. This type of standardization by matrix lines consists of dividing each ROI value by the total addition of ROIs of its same line. As a result,

O. When

H spectra in which

which could simply respond to the urine having a higher or lower concentration of H2

**Figure 3.** Scheme for the construction of a NMR ROI data matrix from 53 NMR spectra. Above: 53 1

*2.4.2.2.1. Standardization method of each ROI line at a standard addition*

the standardized ROIs of a sample add up to the unit.

data matrix obtained through the integration of each of the ROIs.

66 Osteonecrosis

It is a RANKL monoclonal antibody that is currently still undergoing clinical trials for the treatment of osteoporosis, primary and metastatic bone cancer, giant cell tumor and rheumatoid arthritis [116, 117]. RANKL is necessary for the activation and function of mature osteoclasts [118, 119], which together with osteoprotegerin (OPG), maintains the balance of bone resorption in a healthy state. When an imbalance occurs in the RANKL/OPG ratio, resorption is favored in bone diseases [120, 121].

Denosumab has a high specificity due to human IgG2 that binds specifically to human RAN, and not other members of the TNF superfamily [116, 122, 123]. In clinical trials, this drug causes rapid and prolonged decreases in bone exchange markers without any change in bone formation, which gives it antireabsorptives characteristics [124]. It has also shown better clinical results compared to bisphosphonates in the treatment of osteoporosis and cancer with a higher increase in bone density and suppression of bone remodeling markers, with a proven efficacy even in patients who had been previously resistant to bisphosphonates [117, 125, 126].

These drugs also produce osteonecrosis of the jaws with a prevalence of 0.7–19% [127, 128], which is very similar to the osteonecrosis from bisphosphonate treatments [117]. Since the first case of maxillary osteonecrosis due to this drug published in 2010 [129], several studies have been published but only one of them describes histopathologic characteristics [130]. The fragments of necrotic bone showed empty osteocytic lacuna and absence of osteocytes, osteoblasts and osteoclasts. The authors suggest that these characteristics are very similar to bisphosphonate-related osteonecrosis [131]

#### *2.5.2. Antiangiogenics*

These drugs hinder the development of new blood vessels and block the cascade of angiogenesis [132].

**Bevacizumab:** Monoclonal antibodies that stop the growth factor.

**Suntinib and Sorafenib:** Tyrosine kinase inhibitors.
