VEGF- A & B Phase II trial for treatment in. Ovarian, kidney and breast cancer, glioma, Soft tissue sarcoma (STS). (Huang et al.,. 2003). AMG-706. (Motesanib.
1.
INTRODUCTION: ....................................................................................................................... 1 1.1
GROWTH FACTORS: .................................................................................................... 1
1.2
VEGF & ITS RECEPTORS: ............................................................................................. 1
1.3
RATIONALISING VEGF AS A TARGET FOR CANCER TREATMENT: ................................ 3
1.4
AVAILABLE ANTI-VEGF DRUGS: .................................................................................. 3
1.5
VEGF AND VASCULAR HAEMOSTASIS (FIG 2): ............................................................. 5
1.6
ADVERSE EFFECTS OF ANTI-VEGF DRUGS: .................................................................. 7
1.7
POSSIBLE FACTORS CAUSING ANTI-VEGF DRUG INDUCED HYPERTENSION: .............. 8
1.7.1
CHANGES IN ARTERIAL STIFFNESS/MICROCIRCULATION: .......................................... 8
1.7.2
IMPAIRED BARORECEPTOR SENSITIVITY: ................................................................... 9
1.7.3
IMPAIRED AUTONOMIC NERVOUS SYSTEM CONTROL: ........................................... 11
1.7.4
CHANGES IN l-ARGININE/NITRIC OXIDE (NO) PATHWAY: ........................................ 11
1.8
POSSIBLE MECHANISMS CAUSING ANTI-VEGF DRUG INDUCED HYPERTENSION: .... 12
1.8.1
Neuronal pathway: ............................................................................................................. 13
1.8.2
Cardiovascular pathway: .................................................................................................... 13
1.8.3
Chemical pathway: ............................................................................................................. 14
2.
AIMS:...................................................................................................................................... 15
3.
HYPOTHESIS: .......................................................................................................................... 16
4.
MATERIALS & METHODS: ....................................................................................................... 17 4.1
ETHICAL CONSIDERATION: ....................................................................................... 17
4.2
PATIENT SELECTION CRITERIA: ................................................................................. 17
4.3
4.2.1
Inclusion criteria: ............................................................................................................ 17
4.2.2
Exclusion criteria: ........................................................................................................... 18
4.2.3
Withdrawal criteria: ....................................................................................................... 18
CLINICAL EQUIPMENTS USED IN STUDY SITE & THEIR SET UP: ................................. 18 4.3.1
TASK FORCE MONITOR:.............................................................................................. 18
BLOOD PRESSURE MEASUREMENT: ..................................................................................................... 19
1
HEART RATE VARIABILITY: .................................................................................................................... 21 BARORECEPTOR REFLEX SENSITIVITY: .................................................................................................. 21 4.3.2
PULSE TRACE PCA2: .................................................................................................... 21
4.3.3
LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY: ................................. 22
SAMPLE COLLECTION & STORAGE: ...................................................................................................... 23 PREPARATION OF CALIBRATORS AND QUALITY CONTROLS:................................................................ 23 INTERNAL STANDARDS: ........................................................................................................................ 23 ANALYSIS OF SERUM SAMPLES: ........................................................................................................... 23
5.
6.
4.4
CLINICAL PARAMETERS MEASURED: ........................................................................ 25
4.5
STUDY PROTOCOL: ....................................................................................................... 25
4.6
STATISTICAL ANALYSIS: ............................................................................................ 27
RESULTS: ................................................................................................................................ 28 5.1
REPRODUCIBILITY STUDIES ( Table 2): ...................................................................... 28
5.2
PATIENT CHARACTERISTICS (Table 3): ...................................................................... 31
5.3
TREATMENT & STUDY DURATION: ........................................................................... 32
5.4
ADVERSE EVENTS: ..................................................................................................... 32
5.5
STUDY STATISTICS: .................................................................................................... 32
DISCUSSION: ........................................................................................................................... 39 REPRODUCIBILITY STUDIES: ................................................................................................................. 39 BLOOD PRESSURE & ARTERIAL STIFFNESS: .......................................................................................... 40 L-ARGININE: ......................................................................................................................................... 41 NEURONAL FACTORS: .......................................................................................................................... 41 L-ARGININE & ANS FUNCTIONING: ...................................................................................................... 42
7.
CONCLUSION & FUTURE DIRECTIONS: .................................................................................... 44
8.
BIBLIOGRAPHY: ...................................................................................................................... 45
2
3
LIST OF FIGURES Fig 1: Types of VEGF, Receptors & their binding interactions ............................................................. 2 Fig 2: VEGF-VEGFR2 binding induced signals and pathways ............................................................. 6 Fig 3: Cyclic pathway of NO formation and balance .......................................................................... 12 Figure 4: Task Force Monitor Device ................................................................................................. 19 Figure 5: Donning of Continuous Blood pressure device .................................................................... 20 Figure 6: Donning of Oscillometric Blood pressure device ................................................................. 20 Figure 7: PULSE TRACE PCA2 DEVICE ........................................................................................... 22 Figure 8: Schematic representation of proposed mechanism from the study results ........................... 43
LIST OF TABLES Table 1: Anti-VEGF drugs currently in pipeline .................................................................................... 4 Table 2: Reproducibility results ........................................................................................................... 29 Table 3: Patient Demographics ........................................................................................................... 31 4
Table 4: Statistical comparative analysis of patients between baseline and 6 weeks .......................... 34 Table 5: Statistical analysis between hypertension developing patients and hypertension nondeveloping patients............................................................................................................................... 37
5
ABBREVIATIONS:
%
Percentage
°C
Degree Celsius
µl
Microlitre
µmol/L
Micromol/Litre
AAR
Adaptive algorithm
ADAMTS
A Disintegrin And Metalloproteinase with Thrombospondin Motifs
ADMA
Asymmetric Dimethyl Arginine
ANS
Autonomic Nervous System
ARNT
Aryl Hydrocarbon Translocator
BP
Blood Pressure
BRS
Baroreflex Sensitivity
cJUN
Cellular JUN
C-KIT
Stem Cell Factor (CD117 cytokine receptor)
ContBP
Continuous Blood Pressure
CREB
cAMP Response Element Binding
CV
Coefficient of Variation
dBP
Diastolic Blood Pressure
DDAH
Dimethyl hydrolases
DVP
Digital Volume Pulse
ECG
Electro Cardio Graphy
EGFR
Epidermal Growth Factor Receptor
eIF4E1
Eukaryotic Translation Initiation Factor 4E1
eNOS
Endothelial Nitric Oxide Synthase
ERK
Extracellular signal Regulated Kinase
Flk-1
Fetal liver kinase-1
Autoregressive
arginine
parameter
Receptor
Dimethyl
Nuclear
amino
6
Flt-1
Fms like tyrosine-1
Flt-4
Fms like tyrosine-4
FMC
Flinders Medical Centre
g
Gram
GIST
Gastro Intestinal Stromal Tumour
HF
High Frequency
HIF
Hypoxia Inducing Factor
HPLC
High Performance Liquid Chromatography
HR
Heart Rate
HRE
Hypoxia Response Element
HRV
Heart Rate Variability
Hz
Hertz
ICAM
Inter Cellular Adhesion Molecule
ICC
Intraclass Correlation Coefficient
ICG
Impedence Cardio Graphy
iNOS
Inducible Nitric oxide Synthase
IP3
Inositol Triphosphate
IR
InfraRed
JAB1
Jun Activation Domain Binding
KDR
Kinase insert Domain Receptor
LC-MS
Liquid Chromatography Spectrometry
LF
Low Frequency
m/s
Metres/seconds
MAP
Mean Arterial Pressure
MAPK
Mitogen Activated Protein Kinase
MEK
MAPK/ERK Kinase
min
Minute
MKK2
Mitogen activated protein Kinase Kinase 2
–
Mass
7
mm Hg
Millimetre Mercury
MMP
Matrix Metalloproteinase
ms
Milliseconds
mTOR
Mammalian Target of Rapamycin
NF-κB
Nuclear Factor Kappa B
NMMA
N-MonoMethyl L-Arginine
nNOS
Neuronal Nitric Oxide Synthase
NO
Nitric Oxide
NOS
Nitric Oxide Synthase
Nrp
Neuropilin
NSCLC
Non Small Cell Lung Cancer
OscBP
Oscillating Blood Pressure
P value
Probability Value
PCA2
Pulse Contour Analysis 2
PDGFR
Platelet Derived Growth Factor Receptor
PI3K
PhosphoInositol-3 Kinase
PIGF
Placenta Induced Growth Factor
PKB
Protein Kinase B
PKC
Protein Kinase C
PLC
Phospholipase C
PPT
Peak to Peak Time
PSD
Power Spectral Density
RAS
Renin Angiotensin System
RET
Rearranged during Transfection (Tyrosine kinase receptor)
RI
Reflective Index
RVLM
Rostral Ventro Lateral Medulla
S.D
Standard Deviation
S6K
S6 Protein Kinase
8
sBP
Systolic Blood Pressure
SDMA
Symmetric Dimethyl Arginine
SHC
Src Homology 2 Domain Containing
SI
Stiffness Index
STS
Soft Tissue Sarcoma
svVEGF
Snake Venom Derived VEGF
TFM
Task Force Monitor
tPA
Tissue type Plasminogen Activator
tPAI
Tissue type Plasminogen Activator Inhibitor
uPA
Urokinase type Plasminogen Activator
VCAM
Vascular Cellular Adhesion Molecule
VEGF
Vascular Endothelial Growth Factor
VEGFR
Vascular Receptor
Endothelial
Growth
Factor
9
DECLARATION: I certify that this thesis does not contain material which has been accepted for the award of any degree or diploma; and to the best of my knowledge and belief it does not contain any material previously published or written by another person except where due reference is made in the text of this thesis.
Shivshankar Thanigaimani (02/12/2009)
10
ACKNOWLEDGEMENT: I would like to express my utmost gratitude to my supervisor, Dr. Arduino Mangoni for providing me with an opportunity to work on a project which is of significant impact in cancer research. I would like to thank him and Dr. Ganessan Kichenadasse for their support and ideas while clarifying my doubts and leading me in the right path with their valuable comments and suggestions during the course of my project. I thank Dr. Malcolm Whiting, SA pathology for generously providing me with the accessibility and assistance to use Liquid chromatography – Mass spectrometry equipment for my project. I also thank Ms. Lisa, Head Nurse of Haemotology/Oncology day unit for helping me in blood collection with the patients. I take this opportunity to thank my father (C.B. Thanigaimani) and my mother (Lourdes Juliet Thanigaimani) for the immense efforts they had put in for supporting my higher studies which has made me what I am now. I thank them a lot for their support and love. A special thanks to Ms. Saranya Hariharan to be of great emotional support to me during my study period. And many thanks to my friends, Ms. Ramya Thangarajan for being initiative and supportive and Mr. Arpit Dave for his valuable comments and suggestions during my thesis write up.
11
ABSTRACT Anti- vascular endothelial growth factor (VEGF) drugs have evolved as an important class of drugs in cancer treatment of various cancers. These drugs target VEGF, which plays a vital role in vascular homeostasis and this resulted in, various cardiovascular adverse effects associated with its use. Hypertension was the most frequently reported common adverse event with the use of anti-VEGF drugs. The underlying mechanism(s) of anti-VEGF drug induced hypertension has been sparsely studied. The main objective of this study was to identify possible mechanisms for the development of anti-VEGF drug induced hypertension. A laboratory based pilot study was performed to compare the effects of anti-VEGF drugs on various cardiovascular parameters at baseline, 6 weeks and 12 weeks. Since the study is ongoing and also the 12th week measurements are not completed, only the baseline and 6 weeks measurements of 5 patients were available for statistical evaluation. Therefore, parameters were compared and calculated between baseline and 6 weeks. Patients who developed hypertension (Group ‘B’) had an increase in sympathetic tone (20.25 ± 4.87), decrease in vagal tone (-20.75 ± 5.58) and decrease in Larginine levels (-28.5 ± 7.77) at 6 weeks whereas the parameters were unchanged in patients without hypertension (Group ‘A’). Though there were differences between group ‘A’ and ‘B’, there were not statistically significant. There was no significant change in Baroreflex sensitivity and Heart rate and ADMA levels in both the groups. From these results, we speculate that anti-VEGF drugs do not interact with ADMA in short-term; whereas they could cause endothelial dysfunction by decreasing L-
12
arginine bioavailability for NO synthesis and increasing sympathetic tone thereby causing hypertension.
13
1. INTRODUCTION:
1.1 GROWTH FACTORS: Growth factors are proteins capable of stimulating cell growth, proliferation and differentiation. These proteins help in signal regulation during cellular growth in a highly coordinated fashion by binding to their specific receptors (Favoni and de Cupis, 2000). Depending on their structural morphology and physiological function, growth factors are classified into different types. Vascular endothelial growth factor (VEGF) is one of the most important growth factors that regulates angiogenesis during physiological
and pathological conditions and maintains vascular
haemostasis.
1.2 VEGF & ITS RECEPTORS: VEGF family has 7 members identified till date, namely, VEGF – A, B, C, D, E, PIGF (Placenta Induced growth factor) and svVEGF (Snake venom derived VEGF) (Shibuya and Claesson-Welsh, 2006) . VEGF receptors are of 3 major types, namely, VEGFR1 (Flt-1), VEGFR2 (KDR/Flk-1) and VEGFR3 (Flt-4) each being specific to certain types of VEGF ligands (Alitalo and Carmeliet, 2002). In addition, VEGF proteins also bind to Heparin (Ferrara and Henzel, 1989), Neuropilin-1 (Soker et al., 1998) & Neuropilin2 (Gluzman-Poltorak et al., 2000) co-receptors which help in initiation of ligandreceptor binding (Fig 1). 1
VEGFR- 1 & 2 mediate angiogenesis in vascular smooth muscles while VEGFR3 is responsible for lymphangiogenesis (Alitalo and Carmeliet, 2002). VEGF-A ligand and VEGFR-2 interactions are shown to predominantly affect vascular permeability and proliferative functions of the growth factor (Brekken et al., 2000) (Li et al., 2002).
Fig 1: Types of VEGF, Receptors & their binding interactions (uPA – Urokinase plasminogen activator; tPA – Tissue Plasminogen activator; MMP – Matrix metalloproteinase; VEGF – Vascular endothelial growth factor; VEGFR – Vascular endothelial growth factor receptor; Nrp – Neuropilin; PIGF – Placental growth factor) 2
1.3 RATIONALISING
VEGF
AS
A
TARGET
FOR
CANCER
TREATMENT: Angiogenesis is the vital step involved in sprouting of capillaries during cancer progression (Algire, 1945). Clinical and laboratory studies show that VEGF independently interacts and initiates factors that aid in angiogenesis (Ellis, 2007). Because VEGF plays a central role in vascularisation, its inhibition would prevent angiogenic dependent cancer progression. In addition, VEGF is also shown to increase anti-apoptotic proteins (Gerber et al., 1998). Its inhibition would suppress the induction of anti-apoptotic proteins leading to increased cell death or decreased cell survival. These properties of VEGF make it a potential target for cancer treatment and anti-VEGF drugs have shown to decrease disease progression by having a negative effect on tumour growth (Ferrara and Kerbel, 2005).
1.4 AVAILABLE ANTI-VEGF DRUGS: Presently, several Anti-VEGF drugs are approved by various health authorities for treatment in cancer patients e.g., Bevacizumab, Sorafenib and Sunitinib. Among these, Bevacizumab is a humanised monoclonal antibody which is administered intravenously. It specifically binds to VEGF ligand and prevents the VEGF ligand – receptor binding (Kamba and McDonald, 2007). Sunitinib and Sorafenib are multitargeted tyrosine kinase inhibitors which are administered orally (Jain et al., 2006). They target other small tyrosine kinase pathways in addition to VEGF. There are several other anti-VEGF drugs in the pipeline undergoing early phase clinical trials.
3
Table 1: Anti-VEGF drugs currently in pipeline Name of the drug
GW786034B
Target
Currently in
VEGFR1, 2 &3
Reference
Phase III trial for renal cell cancer
(Podar et al., 2006)
(pazopanib)
AZD2171
VEGFR1, 2, 3, Phase II/III trials for various cancers (Goodlad
(Cediranib)
PDGFRβ
and such
as
Breast,
et
kidney, al., 2006)
hepatocellular, ovarian, colorectal
C-KIT
and head and neck cancer, malignant mesothelioma, malignant melanoma, recurrent small cell lung cancer, glioblastoma,
Gastrointestinal
stromal tumour (GIST)
PTK787/ZK2225
VEGFR1, 2, 3, Phase II/III trials for various cancers (Wood et al.,
84 (vatalanib)
PDGFRβ
and such as Colorectal, prostate, renal, 2000)
C-KIT
breast
and
pancreatic
cancer,
glioblastoma, GIST
VEGF trap
VEGF- A & B
Phase II trial for treatment in (Huang et al., Ovarian, kidney and breast cancer, 2003) glioma, Soft tissue sarcoma (STS)
AMG-706
VEGFR1, 2 & Phase II trial for treatment in Non (Polverino et
(Motesanib
3,
PDGFR1 small cell lung cancer (NSCLC), al., 2006)
4
diphosphate)
and KIT
Breast and colorectal cancer
AG013736
VEGFR1, 2, 3 Phase II study for treatment in (Baffert et al.,
(Axitinib)
and PDGFR
pancreatic cancer
ZD6474
VEGFR2,
Phase II trial for treatment in (McCarty
(Zactima,
EGFR, FGFR1 NSCLC, glioma and transitional cell al., 2004)
Vandetanib)
and RET
2005)
et
carcinoma
1.5 VEGF AND VASCULAR HAEMOSTASIS (FIG 2): VEGF, an endothelial cell specific mitogen (Ferrara and DavisSmyth, 1997) is released from normal as well as tumour cells during physiological and pathological angiogenesis (Shibuya, 2008). In addition, pharmacological studies have shown that VEGF is a vasodilator which acts in dose dependent manner in vitro (Ku et al., 1993). It affects the release and activation of various vascular factors that are responsible for maintenance of vascular haemostasis. In addition to induction of angiogenic factors (Shibuya, 2008), VEGF is also responsible for induction and maintenance of several cellular factors and functions. VEGF triggers several cascading signals by binding to their specific receptors. VEGF - VEGFR2 binding triggers various cell signals, predominantly, PI3-AKT (Gelinas et al., 2002), MAPK and Ras-Raf pathways (Fujita et al., 2005). These signals in turn induce several factors such as anti-Apoptotic proteins (Bcl-2 & A1) (Gerber et al., 1998), Endothelial Nitric oxide synthase (eNOS) (Ashrafpour et al., 2004), Inducible Nitric oxide synthase (iNOS) (Zhang and Peng, 2009), Nitric oxide
5
(NO) (Gelinas et al., 2002), Vascular cellular adhesion molecule (VCAM), Intercellular adhesion molecule (ICAM), E-Selectin (Kim et al., 2001), Tissue factor (Senger, 1983), Urokinase type plasminogen activator (uPA) (Mandriota et al., 1995), Tissue type plasminogen activator (tPA) and tPA inhibitor (tPAI) (Pepper et al., 1991), Collagenase (Unemori et al., 1992), Angiotensin-II (Fujita et al., 2005), Endothelin (Shimojo et al., 2007) and Hypoxia inducible factor (HIF) proteins (Ivan et al., 2001). The above discussed VEGF induced factors are responsible for vascular cell proliferation, adhesion, migration, differentiation and pressure responses thereby contributing to vascular haemostasis.
Fig 2: VEGF-VEGFR2 binding induced signals and pathways 6
(PI3K – Phosphoinositol 3 kinase; PKB – Protein kinase B; mTOR – Mammalian target of Rapamycin; S6K – S6 protein kinase; eIF4E1 – Eukaryotic translation initiation factor 4E1; HIF1 α – Hypoxia inducing factor 1α; HIF1 β – Hypoxia inducing factor 1β; ARNT – Aryl hydrocarbon receptor nuclear translocator; CREB – cAMP response element binding; JAB1 – Jun activation domain binding; cJUN – Cellular JUN; HRE – Hypoxia response elements; NF-κB – Nuclear Factor Kappa B; NO – Nitric oxide; NOS – Nitric oxide synthase; ERK – Extracellular signal regulated kinase; PLC – Phospholipase C; PKC – Protein kinase C; ADAMTS – A disintegrin and metalloproteinase with thrombospondin motifs; IP3 – Inositol triphosphate; SHC – Src homology 2 Domain containing; RAS – Renin Angiotensin system; MKK2 – Mitogen activated protein kinase kinase 2; MAPK – Mitogen activated protein kinase; PA – Plasminogen activator; PAI – Plasminogen activator inhibitor; MEK – MAPK/ERK kinase; VCAM – Vascular cellular adhesion molecule; ICAM – Intercellular adhesion molecule) Inhibition of VEGF by anti-VEGF drugs leads to inhibition of such VEGF induced factors and this possibly disturbs vascular haemostasis and normal physiological angiogenesis such as wound healing suggesting that anti-VEGF drugs would affect vascular haemostasis of patient during treatment. Disturbance in vascular haemostasis due to anti-VEGF drugs would be reflected as their vascular related adverse effects.
1.6 ADVERSE EFFECTS OF ANTI-VEGF DRUGS: Anti-VEGF drugs, in addition to their therapeutic effect of reducing tumour growth, cause vascular related adverse effects as a class effect. Such adverse effects of antiVEGF therapy are mainly due to VEGF interaction with several vascular related factor. Hypertension is one of the most common adverse effects observed in patients undergoing anti-VEGF therapy (Sica, 2006). Other adverse events include proteinuria, haemorrhage, thrombosis, impaired wound healing, gastrointestinal perforation, reversible posterior leukoencephalopathy, cardiac impairment and endocrine dysfunction.
7
1.7 POSSIBLE FACTORS CAUSING ANTI-VEGF DRUG INDUCED HYPERTENSION: The mechanism involved in anti-VEGF drug induced hypertension is largely unknown because VEGF interacts with several different factors and signals. Possible responsible factors for anti-VEGF drug induced hypertension are due to the changes in normal homeostasis of blood pressure control.
1.7.1
CHANGES IN ARTERIAL STIFFNESS/MICROCIRCULATION:
Arterial blood vessel wall is the primary site for most cardiovascular diseases (Cohn, 2006). Elastic property of arterial wall transforms pulsatile blood flow from heart into less pulsatile flow when blood reaches distal regions (Safar, 2004). This elasticity of arterial walls protects peripheral microcirculation from pressure induced damage. Therefore, elasticity of arterial walls measured with pulse wave velocity could be used as marker for normal physiological microcirculation as well as other cardiovascular functions (Sutton-Tyrrell et al., 2005). It varies depending on age and blood pressure and several other factors (Millasseau et al., 2002). It is measured from the time taken for pressure waves to travel from an arterial location, usually aortic arch to the site of reflection at the periphery which is relative to height of the subject. Determination of pulse wave velocity is the standard method for measuring arterial stiffness (O’Rourke., 1996). It is calculated as, Stiffness index = Height of the subject / Peak to Peak time (ms) 8
Peak to Peak time is the time taken between obtaining peaks of systolic and diastolic components of waveform. Stiffer the artery, faster will be the reflected wave resulting in decreased peak to peak time. Reflective index is the relative height of diastolic to systolic peak obtained in total amplitude of pulse waveform expressed in percentage. In vascular terms, it is the total amount of blood reflected back from the lower body which is relative to the tone of small arteries (Chowienczyk et al., 1999). It mostly depends on cardiac output, exercise, food intake and also ultimately on the large artery stiffness which is indicated as stiffness index. Therefore, stiffness and reflective index are relative to each other. Stiffness and tone of the artery can be determined by digital volume pulse (DVP) (Millasseau et al., 2002). Any change in the characteristic of DVP would reflect in change in stiffness and reflective index of the artery which would serve as an indicator for development of vascular diseases
1.7.2
IMPAIRED BARORECEPTOR SENSITIVITY:
Baroreceptors are mechanoreceptors located at the inner walls of blood vessels (Paintal., 1972). These receptors are sensitive to change in blood pressure and act by increasing or decreasing the activation of vasomotor centres. They exert relevant effects by responding to circumferential or longitudinal stretch of blood vessels (Heymans, 1958). By relaxing the arterial walls at high blood pressures, these receptors reduce the activation of vasomotor centre and increase the activation of these centres by stretching the walls to limit blood pressure levels within the threshold (Angell James, 1971). The receptor sensitivity to pressure change is more 9
at the lower end of high distensibility region (60-100 mm Hg), i.e., low blood pressure and minimal above the low distensibility region (Above 120-140 mm Hg), i.e., High blood pressure (Kirchheim, 1976). This indicates that baroreceptors are more sensitive to fall in BP rather than its increase. The decrease in sensitivity of receptors is due to loss of arterial distensibility. This is caused by ageing (Franchini et al., 1996), vascular compliance (Kingwell et al., 1995) and size of input signals (Yamazaki and Sagawa, 1989). Baroreceptor mediated vasodepressor responses are controlled by C1 epinephrine neurons in rostral ventrolateral medulla (RVLM) part of brain stem (Granata et al., 1985). Therefore, abnormal signaling from this region might also lead to altered baroreceptor reflexes. Higher the blood pressure, lesser will be the sensitivity and higher will be the threshold limit depending on the period for which high blood pressure persisted (McCubbiin, 1956). An increase in blood pressure is reflected in activation of baroreceptor afferent fibre activation followed by parasympathetic activation and inhibition of sympathetic tone subsequently decreasing heart rate, vascular resistance and venous return (Pang, 2001). In case of chronic hypertension, it is shown that baroreceptor reflexes are reset to higher levels. This is the reason for higher blood pressure levels of patients even after the removal of original cause of hypertension (McCubbiin, 1956). Age and higher blood pressure are shown to decrease baroreflex sensitivity (Gribbin et al., 1971). Vascular stiffness is relative to age as well as hypertension which are maintained by baroreceptor sensitivity (Aars, 1969). Therefore, both vascular stiffness and baroreceptor sensitivity could be relative to each other.
10
1.7.3
IMPAIRED AUTONOMIC NERVOUS SYSTEM CONTRO L:
Heart rate variability is an independent predictor for mortality in acute myocardial infarction (Kleiger et al., 1987). It has a potential to provide insights into physiological and pathological conditions in addition to improve risk stratification (Malik, 1996). It is reflective of autonomic nervous system which is composed of sympathetic and vagal tone. Power spectral density (PSD) analysis can be used to determine autonomic function through transmission of electric signals to a function of frequency. Low frequency which ranges between 0.04 – 0.15 Hz represents sympathetic tone. Vagal tone is represented as high frequency ranging between 0.150.4 Hz (Akinci, 1993). The rate of change of frequency in PSD analysis is in accordance with autonomic modulations of heart rate. It should be noted that HRV denotes only the modulations of autonomic tone and not the actual autonomic levels. The ratio of LF and HF components obtained from PSD indicate the sympatho-vagal balance of the individual.
1.7.4
CHANGES IN L-ARGININE/NITRIC OXIDE (NO) PATHWAY:
Arginine is the sole substrate for NO formation (Moncada et al., 1989). Arginine gets oxidised to NO and citrulline which is catalysed by NO synthase (Wu, 1998). There are 3 isoforms of NO, namely, Inducible (iNOS), endothelial (eNOS) and neuronal (nNOS). This formation of NO is inhibited by methlyarginines such as asymmetric dimethyl arginine (ADMA) and N-monomethyl L-arginine (NMMA) (Alderton et al., 2001). These methylarginines are metabolised by Dimethyl arginine dimethyl amino hydrolases (DDAH) into citrulline and dimethylamine (Ogawa et al., 1989). Nitric oxide inhibits DDAH by replacing its active cysteine residue with serine
11
rendering it inactive (Leiper et al., 2002). These inhibitory and metabolic functions of NO, DDAH, ADMA and NOS occurs in a cyclic manner thereby maintaining haemostatic levels of NO which in turn is responsible for regulation of vascular tone and organ perfusion (Fig 3). Increased pulmonary hypertension leads to higher levels of ADMA (Arrigoni et al., 2003). There are chances of correlation between VEGF and ADMA since both the factors affect NO production effectively.
Fig 3: Cyclic pathway of NO formation and balance
1.8 POSSIBLE
MECHANISMS
CAUSING
ANTI-VEGF
DRUG
INDUCED HYPERTENSION: The pathogenesis of anti-VEGF drug induced hypertension is not thoroughly understood. There are few hypotheses on mechanism of anti-VEGF induced hypertension. The most widely speculated and reasonably acceptable mechanism is that anti-VEGF drugs affect microcirculation resulting in an increased systemic resistance thereby increasing blood pressure. Other possible pathways through which VEGF could affect endothelial function to cause hypertension are,
12
1.8.1
NEURONAL PATHWAY:
Baroreceptor sensitivity and heart rate variability are two factors that could be responsible for anti-VEGF induced hypertension. Baroreceptors send neuronal messages to rostral ventrolateral medulla (RVLM) of central nervous system during alterations in blood pressure and reflect back by exerting an elasticity action on arterial walls (Kubo et al., 1998). Anti-VEGF drugs, by decreasing eNOS synthesis and NO production could cause endothelial dysfunction. This could impair the Baroreceptor functioning as they are present on the inner walls of endothelial cells. This results in decreased signaling capability of baroreceptors to central nervous system. Heart rate variability is indicated as ANS function. An increase in sympathetic tone leads to increase in cardiac output, systemic peripheral resistance and heart rate (Caliva, 1959). Since ANS function is involved in development of hypertension (Schroeder et al., 2003), anti-VEGF drugs could have possible correlation with sympathetic tone in development of hypertension by increasing peripheral resistance.
1.8.2
CARDIOVASCULAR PATHWAY:
Anti-VEGF drugs could increase vascular resistance which should be overcome with an increase in blood flow to maintain a gradual increase in blood pressure and avoid a sudden increase. The blood flow depends on length, diameter and elasticity of blood vessel. These factors are collectively determined as stiffness index. Increased blood flow during increased stiffness index would affect microcirculation. This leads to a decreased peripheral vascular density due to damage to arterioles and capillaries
13
which certainly would increase the speed of reflected wave. Therefore, anti-VEGF could increase the stiffness and reflective index to cause hypertension.
1.8.3
CHEMICAL PATHWAY:
VEGF increase NOS release and NO production through which endothelial cells exert their function. In addition, it is a specific endothelial cell mitogen. Therefore, inhibition of VEGF action by anti-VEGF drugs would affect the normal physiology of VEGF related endothelial cell function. The interaction between VEGF and ADMA is not clearly known. Both VEGF and ADMA are shown to affect endothelial NO function independently. Therefore, AntiVEGF might lead to cardiovascular effects in correlation with ADMA pathway. To date, there are no studies correlating the effects of NO inhibitors and VEGF. VEGF is demonstrated to up regulate eNOS leading to up regulated NO production (Hood et al., 1998) through PI3K/Akt and MAPK pathways (Gelinas et al., 2002). It induces NO dependent relaxation in coronary arteries (Ku et al., 1993) whereas ADMA inhibits NO synthesis and endothelium dependent relaxation of blood vessels (MacAllister et al., 1994). Since both the factors affect NO synthesis, the mechanism in which they act to do so could relate to each other. Therefore, arginine/NO pathway could be a potential mechanism in anti-VEGF induced hypertension. Determination of simultaneous effects of the above mentioned factors during antiVEGF therapy would help in understanding the underlying mechanism involved in anti-VEGF induced hypertension.
14
2. AIMS: To determine the relationship between anti-VEGF drugs and ADMA/NO pathway in increasing blood pressure. To determine if anti-VEGF drugs could cause - Increase in Sympathetic activity, - Impairment of baroreflex receptor sensitivity, - Increase in arterial stiffness, Independently or in relation with ADMA/NO pathway.
15
3. HYPOTHESIS: We hypothesise that, Adverse effects of anti-VEGF drugs on blood pressure are mediated by the ADMA/NO pathway. Anti-VEGF drugs causes increased arterial stiffness and sympathetic tone and impaired baroreflex sensitivity.
16
4. MATERIALS & METHODS:
4.1 ETHICAL CONSIDERATION: This study was prospectively approved by the Flinders Clinical Research Ethics Committee, Flinders Medical Centre, Bedford Park, South Australia. All information obtained from the patients (Initials, Date of birth, contact details, etc.) were coded to maintain confidentiality. The codes were used only for study related purposes. The coded details were maintained to avoid reference to subject’s name. All patients were recruited from the Flinders Cancer Clinic and Haematology-Oncology Day Unit of Flinders Medical Centre (FMC) after signing an ‘Informed consent’. The study was conducted in the department of Clinical Pharmacology, FMC, between March 2009 and November 2009.
4.2 PATIENT SELECTION CRITERIA: Patients with histologically confirmed cancer and planned to be treated with antiVEGF drugs fulfilling the study criteria were approached for their participation in the project.
4.2.1
INCLUSION CRITERIA:
Previously diagnosed cancer patients who were prescribed to begin with antiVEGF therapy either as a sole therapy or in combination with chemotherapy. Patients should not have had anti-VEGF drugs in their previous medication history 17
Should be able to understand, follow the protocol and give informed consent.
4.2.2
EXCLUSION CRITERIA:
Poorly controlled hypertension Chronic Grade 4 renal failure / coronary artery disease / liver dysfunction / dementia / previous thromboembolism. Patients already being treated with anti-VEGF therapy. Unable to comply with protocol.
4.2.3
WITHDRAWAL CRITERIA:
Cessation of therapy within 12 weeks.
4.3 CLINICAL EQUIPMENTS USED IN STUDY SITE & THEIR SET UP:
4.3.1
TASK FORCE MONITOR:
Task force monitor is a portable diagnosis support device which can be used for noninvasive long term monitoring of patients’ haemodynamic parameters such as Impedence cardiography (ICG), Electro cardiography (ECG), Oscillometric (OscBP) and Continuous (ContBP) blood pressure measurement, Heart rate variability and baroreceptor sensitivity with beat to beat statistics (Fortin, 1998). The device provides electric signals relating to haemodynamic parameters but without any diagnostic purposes.
18
Figure 4: Task Force Monitor Device (Red – ECG port; Silver – Air supply for Continuous and Oscillometric blood pressure device port; Green – Continuous blood pressure device port.) In this study we used TFM in real time analysis of blood pressure, heart rate variability along with baroreflex receptor sensitivity.
BLOOD PRESSURE MEASUREMENT:
Blood pressure was measured in two forms as continuous and oscillometric BP. The continuous BP device consists of: 1.
Flying ‘V’ cuff,
2.
Vascular unloading monitor,
3.
Removable forearm fixing cuff and
19
4.
A compressed air hose.
The continuous BP cuff was placed in the two fingers as shown in Figure 5. Poor signals were experienced during measurement which was corrected by warming the hand with warm water and/or by switching the fingers of measurement in continuous BP cuff.
Figure 5: Donning of Continuous Blood pressure device An upper arm blood pressure cuff was used to measure oscillometric BP. The cuff was placed such that the air outlet was in front of brachial artery and just above the elbow to ensure precise measurement as shown in Figure 6.
Figure 6: Donning of Oscillometric Blood pressure device
20
HEART RATE VARIABILITY:
The power spectral analysis of TFM uses an adaptive autoregressive parameter (AAR) algorithm to determine the heart rate variability (Schlogl A, 1997). The frequency of RR interval to beat-to-beat tachogram signal were measured and converted into electric signals which was displayed on the monitor (Bianchi, 1997).
BARORECEPTOR REFLEX SENSITIVITY:
The spontaneous baroreceptor activity within the cardiovascular system is determined by baroreceptors and sent to brain stem. This spontaneous activity of the individual is determined by TFM through sequence method which analyses and displays the total number of rising and falling sequences during monitoring (Parati G, 1992). The device shows a pattern of BP – Up and Down events.
4.3.2
PULSE TRACE PCA2:
Pulse trace Pulse Contour Analysis (PCA2) is a battery operated portable digital volume pulse measurement system. It uses photo plethysmography transducer to obtain indices related to stiffness and vascular tone of artery.
21
Figure 7: PULSE TRACE PCA2 DEVICE Photo plethysmography works on the principle that transmission of IR light at 940nm through the finger is proportional to volume of blood in the finger (Chowienczyk et al., 1999). The amount of light that is able to transmit through the finger gives a digital volume pulse on the screen which appears as a waveform. The probe was placed in the ring finger of the patients and the spot check which displays the real time pulse volume waveform was used to determine the arterial stiffness. The patients were requested to be studied in the morning or avoid excessive exercise/caffeine/smoking/alcohol for the preceding 24 hours. Patients were rested for at least 15 min prior to the start of protocol to attain a haemodynamically stable condition.
4.3.3
LIQUID CHROMATOGRAPHY TANDEM MASS SPECTROMETRY:
Mass spectrometry (MS) is an analytical technique that provides both qualitative and quantitative information of ionised molecules. When high performance liquid chromatography (HPLC) is coupled with MS, it fractionates the molecules according to their size and polarity. Therefore, large and small molecules can be fractionated prior to mass spectrometric analysis which renders more sensitive and reliable result. This application was used in this study to determine the serum concentration of NO inhibitors and their substrate. The serum NO inhibitor levels were determined with liquid chromatography-mass spectrometry (LC-MS) by following the standard protocol (Schwedhelm et al., 2005) with the help of SA pathology.
22
SAMPLE COLLECTION & STORAGE:
Three sets of blood samples (Baseline, 6 weeks & 12 weeks) were collected for each study participant in a serum separator activator tube. The collected blood samples were centrifuged at 5000g for 5 min to sediment the blood cells. The serum remains on the top of the gel portion in the serum separator activator tube. The separated serum was transferred to a previously coded tube and frozen at -20°C.
PREPARATION OF CALIBRATORS AND QUALITY CONTROLS:
Three different calibrators were used (ADMA, SDMA and Arginine) at six different concentrations: 0.1, 0.25, 0.5, 1 and 2 µmol/L concentrations respectively.
INTERNAL STANDARDS:
Deuterated ADMA and Arginine were used as internal standards at 10 and 100 µmol/L concentrations respectively in the form of deuterated mix.
ANALYSIS OF SERUM SAMPLES:
20µl of deuterated mix (Internal standard) was added to each sample, calibrators and quality controls. 100µl of acetone was added to precipitate the proteins. The precipitated proteins were centrifuged at 10,000g for 10 min to sediment the precipitated proteins. 100µl of supernatant was transferred to another lidless eppendorf. The supernatant was left to dry over 2 days. 75µl of derivatising agent was added to the dry supernatant and incubated at 65°C for 17 min. The samples were evaporated to dry under nitrogen to eliminate traces of moisture content. The
23
samples were reconstituted with 200µl of water and vortexed. 180µl of sample were added to microtitre plate and placed in LC-MS which collects approximately 20-25µl for determining the concentrations of L-Arginine, ADMA and SDMA.
24
4.4 CLINICAL PARAMETERS MEASURED:
TASK FORCE
PULSE TRACE PCA2
LC-MS
Heart rate (HR)
ADMA
Stiffness Index (SI)
SDMA
Reflective Index (RI)
L-Arginine
Peak to Peak time (PPT)
--------------------------
Low Frequency (LF)
---------------------------
--------------------------
High Frequency (HF)
----------------------------
---------------------------
LF/HF
----------------------------
---------------------------
MONITOR
Systolic Blood pressure (sBP)
Diastolic Blood pressure (dBP)
Mean Arterial pressure (MAP)
Baroreflex sensitivity (BRS)
4.5 STUDY PROTOCOL: 25
This is a laboratory based clinical study in which vascular related haemodynamic parameters were studied to determine their relationship with anti-VEGF induced hypertension. The study was designed for 30 min. Skin area was disinfected before donning the electrodes. The patients were equipped with 4 ECG electrodes and the cable was plugged into red ECG panel jack of TFM. The upper arm BP cuff was wrapped on the left hand of the patients. The air hose of upper arm cuff was connected to oscillometric connector of TFM. A continuous BP finger ‘V’cuff was placed in the middle and index finger of the right hand to measure continuous blood pressure. The ‘V’ cuff was available in three different sizes (small, medium and large) which was appropriately checked with the subject’s finger diameter before use. A probe connected from Pulse trace unit was placed on the ring finger of right hand in such a way that the edge of the finger touches the ledge of the probe and the cable was coming from the ventral part of the palm. Patients were rested in supine position for first 10 min to attain haemodynamically stable condition. Clinical parameters were measured for the following 20 min using Task Force Monitor (TFM, CNSystems Medizintechnik AG, Graz, Austria) and pulse trace PCA2 (Micro medical limited, Rochester, England). After measuring the clinical parameters, approximately 5ml of blood was collected from patients in a serum separator activator tube for further serum 26
analysis using liquid chromatography tandem mass spectrometry (SA pathology, Adelaide, Australia). Clinical parameters were measured and blood samples were collected at baseline of the study before the start of therapy. The protocol was repeated with the patients at 6 weeks and 12 weeks after starting the therapy.
4.6 STATISTICAL ANALYSIS: Coefficient of variation (CV), Intraclass correlation coefficient (ICC) and Paired T – test were calculated using SPSS and Sigmastat statistical software for analyzing the data.
27
5. RESULTS:
5.1 REPRODUCIBILITY STUDIES ( TABLE 2): Healthy volunteers were selected to test the reproducible efficiency of equipments used. The similar protocol designed for patients were performed with 7 volunteers on two different days except that no blood sample was collected from volunteers. Coefficient of Variation was found to be less than 15 % in all parameters except BRS and LF/HF. The variations of all parameters were within the significant range from mean value except BRS and LF/HF. Intraclass Correlation Coefficient ranged between 0.07 to 0.9 for all the measure parameters (Table 2). Overall, statistical results show that the results could be reproduced if the experiment is repeated.
28
Table 2: Reproducibility results
CLINICAL PARAMETERS
COEFFICIENT OF
INTRACLASS
VARIATION (CV)
CORRELATION (ICC)
Systolic blood pressure - SBP 4.5
0.88
6.5
0.85
5.1
0.9
Heart rate - HR (bpm)
5.9
0.82
Peak to Peak time – PPT (ms)
4.6
0.07
Stiffness Index - SI (m/s)
4.9
0.65
Reflective Index – RI (%)
11.8
0.41
20.5
0.36
(mmHg)
Diastolic Blood Pressure DBP (mmHg)
Mean Arterial Pressure - MAP (mmHg)
Baroreflex sensitivity – BRS (mmHg/ms)
29
Low Frequency – LF (%)
10.6
0.77
High Frequency – HF (%)
12.2
0.77
LF/HF (%)
26.4
0.81
30
5.2 PATIENT CHARACTERISTICS (TABLE 3): A total of 6 patients were enrolled in this laboratory based clinical study between March, 2009 till September, 2009. All patients were in their metastatic stage of the disease with an age range of 53 – 69. Five out of 6 patients received Avastin (Bevacizumab) as their anti-VEGF therapy drug. One patient received Pazopanib as their anti-VEGF therapy drug. Patients were already on chemotherapy for cancer treatment before the start of anti-VEGF therapy and continued to take chemotherapy along with anti-VEGF drug. All patients were on anti-hypertensive drug before and during the study. Table 3: Patient Demographics CHARACTERISTICS
NUMBER OF PATIENTS (n=6)
Age (Years)
Range = 53-69
Gender
Male (5), Female (1)
Race
Caucasian (100%)
Prior Chemotherapy
Yes (100%)
Smoking status
Ex smoker (100%)
History of hypertension
Yes (100%)
Renal Failure
No (100%)
Diabetes
3 (50%)
31
Cancer Stage
Metastatic (100%)
Anti-VEGF drug Administered
Avastin (5), Pazopanib (1) 430 – 550 mg of Avastin I.V every two weeks,
Dose of drug administered 400 mg of Pazopanib BD Orally
5.3 TREATMENT & STUDY DURATION: Avastin was administered intravenously for every two weeks. Pazopanib was administered orally twice daily. The anti-VEGF drug was given after the baseline study. The study was designed for 12 weeks with the clinical parameters measured at baseline followed by 6 and 12 weeks. Anti-VEGF therapy was continued in the patients as part of their treatment after the study period.
5.4 ADVERSE EVENTS: One participant on Avastin, developed arterial thrombosis in the infused arm possibly related to anti-VEGF therapy. This participant was withdrawn from further anti-VEGF therapy and from the study according to the withdrawal criteria. Another patient on Pazopanib suffered bleeding due to anti-VEGF therapy and treatment was temporarily stopped for 2 weeks during the interim measurements.
5.5 STUDY STATISTICS: Table 4 shows the MEAN ± S.D of measured clinical parameters of the whole patient group during Baseline and 6 weeks along with ‘P’ value which was calculated by performing ‘PAIRED T TEST’. Twelfth week measurements were not 32
considered for calculation since the study is not yet completed for all patients. Five patients were eligible for statistical analysis to compare the effects of anti-VEGF drugs at baseline and 6 weeks. There was no remarkable change in blood pressure and heart rate at 6 weeks. There was an increase of 1.16 (m/s) in stiffness index and a fall of 14.42 (ms) in peak to peak time without any significant change in reflective index of the whole group. Baroreflex sensitivity was not altered whereas there was a proportional increase of 8.12 (%) in sympathetic tone and decrease of 8.32 (%) in vagal tone and 13.4 (μmol/L) in L-arginine levels. ADMA and SDMA levels did not change significantly in both the groups. There were no statistically significant changes in any of the measured clinical parameters in the whole patient group.
33
Table 4: Statistical comparative analysis of patients between baseline and 6 weeks CLINICAL
BASELINE
PARAMETERS
(N=5) A MEAN (± S.D)
WEEK 6 (N=5)
B
MEAN (±
B-A
‘P’
(Difference
VALUE
in Mean)
S.D) Systolic blood
119.74 ± 12
114.2 ± 13.05
-5.54
0.3665
77.68 ± 13.95
74.26 ± 18.39
-3.42
0.5333
Mean Arterial Pressure 88.06 ± 13.19
84.36 ± 17.37
-3.7
0.5112
42.06 ± 6.07
39.94 ± 7.1
-2.12
0.1294
Heart rate - HR (bpm)
79.36 ± 8.86
75.98 ± 7.74
-3.38
0.2664
Peak to Peak time –
182.54 ± 25.12
168.12
pressure – SBP (mmHg) Diastolic Blood Pressure – DBP (mmHg)
– MAP (mmHg) Pulse Pressure – PP (mmHg)
PPT (ms) Stiffness Index – SI
± -14.42
0.4406
43.04 9.57 ± 0.79
10.73 ± 2.37
1.16
0.3256
65.58 ± 5.58
65.64 ± 8.08
0.06
0.9862
5.95 ± 2
6.23 ± 3.75
0.28
0.8170
(m/s) Reflective Index – RI (%) Baroreflex sensitivity
34
– BRS (mmHg/ms) Low Frequency – LF
51.98 ± 22.91
60.1 ± 19.01
8.12
0.2315
48.22 ± 22.88
39.9 ± 19.01
-8.32
0.2311
LF/HF (%)
1.32 ± 1.02
1.6 ± 1.08
0.28
0.6591
ADMA (μmol/L)
0.63 ± 0.14
0.58 ± 0.15
-0.05
0.4831
SDMA (μmol/L)
0.57 ± 0.17
0.63 ± 0.15
0.06
0.4042
L-Arginine (μmol/L)
150.6 ± 30.21
137.2 ± 20.16
-13.4
0.4872
(%) High Frequency – HF (%)
35
In Table 5, Patients were separated into two groups for comparing the change in clinical parameters between those who developed a significant rise in their blood pressure called the hypertension group (B) in their 6th week of study and those who did not develop hypertension (A). ‘P’ value was not calculated due to smaller sample size in each group. There was an increase of approximately 6 mmHg in sBP, dBP and MAP respectively in hypertension developed patients (Group B) at 6 weeks. Peak to peak time decreased by 21.9 ms in hypertension developed patients and 9.43 ms in nonhypertensive patient group (Group A). There was no significant change in stiffness index of both groups but reflective index increased by 2.4% in only group-B. BRS decreased by 1.05 mmHg/ms in hypertension patient group and increased by 1.17 mmHg/ms in non-hypertensive patient group. But the changes in BRS in both the groups were insignificant. Sympathetic tone increased by 20.22 % and vagal tone decreased by 20.72 % in hypertension patient group whereas there it remained stable in the other group. Heart rate remained stable in all patients of both the groups.
36
Table 5: Statistical analysis between hypertension developing patients and hypertension non-developing patients PATIENTS WITHOUT
PATIENTS WITH
CLINICAL
HYPERTENSION
HYPERTENSION (N
PARAMETERS
(N = 3) A
=2) B
MEAN DIFF ± S.D
MEAN DIFF ± S.D
Systolic blood pressure – -12.73 ± 10.06
5.25 ± 1.48
-10.5 ± 7.87
7.2 ± 2.12
-10.66 ± 9.03
6.75 ± 0.63
-2.23 ± 2.41
-1.94 ± 3.6
-4.43 ± 3.74
-1.79 ± 10.04
-9.43 ± 47.06
-21.9 ± 32.66
0.9 ± 2.73
1.54 ± 2.42
SBP (mmHg) Diastolic Blood Pressure – DBP (mmHg) Mean Arterial PressureMAP (mmHg) Pulse Pressure – PP (mmHg) Heart rate – HR (bpm) Peak to Peak time – PPT (ms) Stiffness Index – SI (m/s)
37
PATIENTS WITHOUT
PATIENTS WITH
CLINICAL
HYPERTENSION
HYPERTENSION (N
PARAMETERS
(N = 3) A
=2) B
MEAN DIFF ± S.D
MEAN DIFF ± S.D
Reflective Index – RI (%)
-1.49 ± 6.71
2.4 ± 10.18
1.17 ± 3.16
-1.05 ± 0.35
Low Frequency – LF (%)
0.03 ± 8.64
20.25 ± 4.87
High Frequency – HF (%)
-0.03 ± 8.64
-20.75 ± 5.58
LF/HF (%)
-0.43 ± 0.80
1.35 ± 1.34
ADMA (μmol/L)
-0.053 ± 0.16
-0.04 ± 0.15
SDMA (μmol/L)
0.106 ± 0.17
-0.015 ± 0.02
L-Arginine (μmol/L)
-3.33 ± 51.59
-28.5 ± 7.77
Baroreflex sensitivity – BRS (mmHg/ms)
38
6. DISCUSSION: In this study, we investigated the simultaneous effects of anti-VEGF drugs on the ADMA/NO pathway as well as established neuronal and cardiovascular factors controlling blood pressure. The primary aim of the study was to identify the mechanisms of anti-VEGF drug induced hypertension by measuring the changes in clinical/laboratory parameters at 6 and 12 weeks of anti-VEGF therapy compared to baseline measurements before the start of therapy.
REPRODUCIBILITY STUDIES:
Initially, reproducibility studies were performed with volunteers. Calculations of the Coefficient of variation (CV) and the Intraclass correlation coefficient (ICC) were carried out to determine the reproducibility of the techniques. CV denotes the dispersion of residuals in each variable from the mean. For most variables tested, CV of less than 15% indicates that residuals of each measured parameters have not varied to a greater extent from mean value. Since the dispersion of residuals is less, it means to be a good model fit. This demonstrates the good reproducibility of the technique. Dispersion of variables was more than 15% for BRS and LF/HF measurements, indicating that the residuals vary in a greater extent compared to the mean. In LF/HF, since the mean was a small number = 1.56 (i.e., nearer to zero), CV varies largely even for a small change in difference between two variables. Therefore, CV cannot be relied in case of variables whose mean is nearer to zero. Lesser the mean, less reliable will be the CV. 39
ICC was calculated to determine the homogeneity of measurement of different variables within the same class. The ICC of a parameter can range between -1 to +1 (McGraw and Wong, 1996). The closer the ICC value to +1 the more reproducible is the measure. Therefore, the results indicate that all parameters had a good reproducibility.
BLOOD PRESSURE & ARTERIAL STIFFNESS:
The results obtained indicate that blood pressure decreased in 3 out of 5 patients. One possible reason is the higher blood pressure values at baseline, which could be due to a psychological effect. Therefore, a significant increase in BP at 6 weeks was not clearly established. Hypertension is generally due to an increase in peripheral resistance (RI) and/or a decrease in arterial compliance (SI). If only peripheral resistance is increased, MAP would increase with a similar proportional increase in both sBP and dBP. However, if arterial compliance is decreased, MAP would increase to the same extent but with a proportionally higher increase in sBP vs. little increase in dBP (Nichols and McDonald, 1972). The patients who developed hypertension at 6 weeks (n = 2) had proportional increase of ~ 6 mmHg in sBP, dBP and MAP respectively compared to patients who did not develop hypertension (n = 3). This suggests that the increase in blood pressure during treatment with anti-VEGF drugs, at least in the short-term, was likely to be secondary to a specific increase in peripheral resistance. This is further supported by decreased peak to peak time and an increased reflective index in
40
hypertension developed patient group, the indices which are affected by an increased peripheral resistance.
L-ARGININE:
Decrease in L-Arginine levels at 6th week of analysis in the whole group indicates that anti-VEGF drugs have a temporal effect on them. In patients who developed hypertension, the decrease was higher which substantiates the effect of anti-VEGF drugs on L-Arginine. Since L-arginine is the sole substrate for NO synthesis (Moncada et al., 1989), its decreased availability would lead to decreased NO production which is the key process for the manifestation of endothelial dysfunction during hypertension (Watson et al., 2008). Decrease in L-arginine levels does not reflect on increased NO synthesis since there is also a proportional increase in sympathetic tone. Several studies (Jimbo., 1994) (Sander et al., 1995) and reviews (Patel et al., 2001) demonstrate an antagonistic relationship between sympathetic nervous system and NO. Thus, it could be accomplished that anti-VEGF drugs decreases L-arginine levels and increases sympathetic tone. This results in increased NO inhibition without any interaction with ADMA pathway as the ADMA levels were unchanged. Alteration of L-arginine levels could be a potential mechanism of anti-VEGF drugs in causing endothelial dysfunction during hypertension.
NEURONAL FACTORS:
An increase in sympathetic tone and relative decrease in vagal tone indicate that antiVEGF drugs affect ANS functioning. There was no significant change in BRS and
41
HR. Increase in sympathetic tone would increase the peripheral resistance leading to increased blood pressure (Caliva, 1959). In order to prevent this increase in blood pressure, BRS should be hyperactive by decreasing the HR (Ernsting, 1957). But the results show that BRS and HR were unchanged with an increase in ANS functioning. Increased blood pressure and shear stress would damage the inner walls of blood vessels resulting in endothelial dysfunction. It could be concluded that anti-VEGF drugs may cause endothelial dysfunction by increasing the sympathetic tone and decreasing vagal tone.
L-ARGININE & ANS FUNCTIONING:
Decrease in L-Arginine levels with a relative increase in sympathetic tone and decrease in vagal tone indicates that these factors may be associated with each other in causing anti-VEGF induced endothelial dysfunction. Previous studies show that L-arginine and sympathetic system has relative effects on vasodepressor actions (Abdelrahman and Pang, 2002). Further studies illustrates the negative regulation of sympathetic system (Nishimura et al., 1997) and facilitating action on vagal tone (Chowdhary et al., 2002) by L-arginine.
42
Figure 8: Schematic representation of proposed mechanism from the study results
43
7. CONCLUSION & FUTURE DIRECTIONS: Our preliminary findings suggest that anti-VEGF drugs do not interact with ADMA pathway but induce hypertension by decreasing L-arginine levels which, in addition to decreasing NO synthesis, increase sympathetic tone thereby initiating anti-VEGF induced endothelial dysfunction. The small sample size and short duration for analysis (6 weeks) were the limitations of the study. Long term studies in a larger sample could confirm this hypothesis.
44
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