This article discusses the causes of hypoxia in various disease states, and the role of hypoxia in the development of diagnostic imaging agents and novel treatments
A variety of pathological conditions exist where the affected tissues are hypoxic or exhibit a low oxygen tension. Primarily, the therapeutic consequences of hypoxia have been considered in the treatment of cancer. However, it is also an important factor where an inflammatory component exists, eg, chronic inflammatory bowel disease, rheumatoid arthritis and ischaemia/reperfusion injury. Moreover, hypoxia must be considered as a factor in diseases where oxygen tensions are likely to be insufficient due to poor respiratory function, eg, cystic fibrosis and chronic bronchitis, as well as to benign proliferative diseases like psoriasis, diabetic vasculopathies and epilepsy.
Advances in research have yielded a means of exploiting this feature, both as a means of imaging and diagnosing disease, as well as for therapy. Examples of therapeutic approaches that offer a means of specifically treating these conditions include the use of bioreductive drugs (ie, prodrugs that are activated only under conditions of hypoxia), gene therapy, and inhibitors of enzymes crucial to the survival of hypoxic cells. This article will discuss the causes of hypoxia in various disease states, and its role in the development of diagnostic imaging agents and novel treatments.
Cells become hypoxic when, for a variety of reasons, oxygen demand exceeds oxygen supply. Hypoxia arises in solid tumours due to the rate of proliferation of their component cells. In normal tissue, an adequate oxygen and nutrient supply is maintained by structurally and functionally normal vasculature, which is able to expand and adapt to meet the needs of the growing tissue. Some tissues, such as skin and cartilage, are slightly less oxygenated. However, as a rule, normal tissues are sufficiently well vascularised to provide a partial oxygen pressure that exceeds 40mmHg.1 Figure 1 shows the range of partial oxygen pressures occurring in solid tumours as compared with various organs. An aggressively growing tumour will depend upon host tissue derived vascularisation. This vasculature is only sufficient to support selected parts of the tumour for a relatively short time, and as the tumour progresses, morphological and functional changes take place. Hypoxia within the tumour stimulates the production of vascular endothelial growth factor (VEGF), which in turn gives rise to tumour-derived angiogenesis.2,3 However, tumour derived vasculature is distinct from that of normal tissue in that vessels are unevenly distributed throughout the tumour, leading to avascular areas. In addition, tumour blood supply is further compromised as those vessels contained within the tumour are often elongated, dilated and twisted. These morphological changes, together with the accompanying rheological changes that occur as a result of the destabilisation of blood supply, can cause an increase in interstitial pressure, which further decreases perfusion.
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Figure 1: Oxygen tension in solid tumours and normal tissues (adapted from reference 6) |
Figure 2: Factors that determine the pO2 distribution in tumour tissue (adapted from reference 4) |
Tumour hypoxia can occur in a chronic or acute form. The location, extent and the relative number of acutely or chronically hypoxic cells vary within and between individual tumours. However, in both chronic and acute hypoxia, oxygen deficiency initially develops in tissue distant from a functional tumour capillary and to the greatest extent at the venous end of the microvessel.5 A solid tumour that is chronically hypoxic shows a characteristic pattern (see Figure 3), whereby proliferating cells are situated at close proximity to a functional blood vessel and necrotic cells are situated in an outer layer. The cells that lie between the inner healthy cells and the outer, necrotic cells are chronically hypoxic and phenotypically adapted to these conditions.6 These cells are continually replaced by cells that are displaced from their position nearest to the blood vessel,7 and, in a variety of human and mouse tumours, have been found to lie in a layer situated at a distance of between 50 and 250µm.8 Anoxia, ie, a complete lack of oxygen, occurs in tumours with a radius in the region of 145µm or larger.1
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Figure 3: Pattern of viable, hypoxic and necrotic cells in a solid tumour (taken from reference 5, with permission) |
Cells exposed to a hypoxic environment are radio- and chemo-resistant. These resistant cells can remain viable after treatment, leading to the possibility of recurrence and metastasis on cessation of therapy. In addition, the adaptations necessary to maintain viability of these cells in hypoxic conditions may cultivate a more malignant phenotype - there is some evidence that hypoxia favours selection of aberrant p53 tumour suppresser gene9 and that exposure to hypoxia increases tumour growth rate and experimental metastatic potential.10 A correlation has also been found between hypoxia-induced tumour-derived angiogenesis, quantified by means of VEGF-induced areas of dense vascularisation, or "vascular hot spots", and poorer five-year survival and increased local recurrence in patients with cervical tumours.11 Cells cultured in anoxic conditions, ie, in the absence of oxygen, are approximately three times more resistant to the cytotoxic effects of radiation than fully aerated cells, radiosensitivity rapidly increasing with increasing oxygen concentration. The term used to express this effect is the oxygen enhancement ratio (OER), which is the ratio of radiation doses under hypoxic versus aerobic conditions to produce a similar biological effect.
Radioresistance occurs due to the deficiency of molecular oxygen, which acts as a radiosensitiser due to its action as an electron acceptor. As a consequence, the free radical formation that results in cell damage occurs to a much lesser extent under hypoxic conditions, leading to survival of these dangerous cells. Hypoxia also confers changes in radiosensitivity due to the proliferative, physiologic and metabolic changes associated with cells exposed to this adverse microenvironment, eg, DNA repair following radiotherapy may be augmented.12
Viable hypoxic cells possess certain characteristics that enable them to survive the adverse conditions in which they are placed. The presence of hypoxia in mammalian tissue effects systemic and metabolic responses, such as increased ventilation, cardiac output and haemopoiesis, as well as biochemical changes, including an increase in anaerobic glycolysis and the level of protective stress proteins.
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Figure 4: Binding of HIF-1 heterodimer to the 5' TACGTTGCT 3' hypoxia responsive element (HRE) can only take place upon dimerisation of HIF-1a and HIF-1b (ARNT) (adapted from reference 20) |
The association between hypoxia and inflammation is well established, and experimental models of ischaemia and reperfusion injury have enabled the elucidation of hypoxia regulated expression of certain inflammatory mediators. Reperfusion injury is mediated by the accumulation of hypoxanthine and xanthine oxidase during ischaemic episodes, which occur as a result of the conversion of xanthine dehydrogenase to xanthine oxidase. The metabolism of hypoxanthine to xanthine by xanthine oxidase is oxygen dependent. Therefore, sufficient metabolism is hindered in ischaemic conditions. When reperfusion takes place, and normal oxygen tensions resume, xanthine oxidase is able to convert the accumulated hypoxanthine to xanthine with the generation of large amounts of superoxide ion, H2O2 and other oxygen radicals. These metabolites initiate lipid peroxidation of the cell membranes with the subsequent release of chemotactic agents, including arachidonic acid products such as leukotriene B4, thromboxane A2, and the interleukins IL-1 and IL-8. These chemotactic agents play a significant part in the migration, adhesion and activation of neutrophils, which are key players in the inflammatory process and have been implicated in the resulting tissue injury.23–25
The precise complement of chemotactic agents, and their regulation, is not fully understood. Hypoxia profoundly affects production of IL-2, IL-4 and interferon gamma (IFNg), but decreases the production of IL-10 by human monocytes.26 It is also known that hypoxia induces the production of the chemotactic agents IL-8, IL-1 and active IL-1a by macrophages and endothelial cells, both at the mRNA and the protein level. However, the duration of hypoxia, and the need for reoxygenation to achieve up-regulation and the downstream effects of these agents varies according to the experimental model that has been used.27–30 In addition, it must also be considered that hypoxia induces production of IL-6, which has been shown to possess anti-inflammatory properties such as the suppression of IL-1.31 Therefore, it could be said that the extent of inflammation is, in part, dependent upon the balance of the effects of hypoxic stimulation of particular interleukins.
Vasodilatation and increased vascular permeability accompany inflammation, which is a way of ensuring delivery of various leukocytes and their products. Important vasoactive substances include nitric oxide and the prostaglandins, production being mediated by the action of nitric oxide synthases (NOS) and cyclo-oxygenases (COX-1 and COX-2), respectively. Although it is known that hypoxia induces expression of COX-232 and is associated with maintenance of prostaglandin E2 in rheumatoid synovial fibroblasts,33 its effect on NOS expression is controversial. Unfortunately, different experimental models have provided conflicting results. One form of the enzyme, known as iNOS, is inducible by inflammatory mediators such as the interleukins, which are in turn hypoxia inducible. Endothelial nitric oxide synthase (eNOS) is also indirectly induced in hypoxic conditions, via an increase in intracellular calcium ions, which are involved in the activation of the enzyme.34
It is well established that polymorphonuclear leukocytes such as neutrophils have a detrimental, rather than the initially suggested beneficial effect on healing post myocardial infarction. Neutrophil-mediated endothelial injury takes place when the neutrophil binds via adhesion receptors to endothelial ligands such as the E-selectins (ELAM-1, CD62E) and leukocyte integrins such as ICAM-1, ICAM-2 and LFA-1. Cultured endothelial and muscle cells increase their adhesiveness for neutrophils under hypoxic conditions via an LFA-1 dependant mechanism.35 Further work has identified the existence of an adhesion ligand detectable by an antibody with affinity to an endothelial cell surface ligand termed HAL 1/13. Although there is no increase in HAL 1/13 density in response to hypoxia, the functionality of the molecule, ie, its contribution to leukocyte adherence, increases significantly in these conditions.36 The contribution of ICAM-1 and ICAM-2 to hypoxia-induced leukocyte adhesion is controversial. Whereas some studies have failed to find significant involvement,36,37 further work has demonstrated that a combination of hypoxia and lipopolysaccharide acts as a stimulus for ICAM-1 expression and ICAM-1 mediated leukocyte adhesion. However, this effect is not universal, occurring concomitantly with hypoxia induced expression of the nuclear transcription factor NF-kB.38 Anti-IL-1 antibodies suppress hypoxia induced leukocyte adherence, indicating a central role for IL-1.27 In addition, it has been demonstrated that hypoxia-induced IL-1 release leads to a reperfusion linked autocrine enhancement in the expression of adhesion molecules such as ICAM-1.30 E-Selectin production is stimulated in aortic endothelial cells by a combination of hypoxia and lipopolysaccharide, or hypoxia and tumour necrosis factor (TNF) possibly via a hypoxia induced decrease in c-AMP production.39 This is of particular significance, as it is has been shown that ischaemia stimulates TNF production in hepatic reperfusion injury.40
The activation of the neutrophil initiates the release of proteolytic enzymes and reactive oxygen metabolites into the extracellular space, culminating in the lysis of essential structural matrix proteins, increased microvascular permeability and oedema.23 Neutrophil-mediated tissue damage is limited during inflammation by apoptosis, or the programmed death of these cells. Hypoxia prolongs the survival of neutrophils,41 and has also been used experimentally to derive a population of macrophages that are also resistant to apoptosis, and will therefore perpetuate the production of inflammatory mediators, and conversely, the activation of these resistant neutrophils.42
The outcome after a myocardial infarction is profoundly limited by the incidence of reperfusion injury, increasing the chance of arrhythmias, myocardial stunning and infarct size.43 Paradoxically, hypoxia regulates growth, proliferative capacity and collagen type-1 production in cardiac fibroblasts, and therefore may play a part in the post-infarct remodelling of the cardiac collagen matrix.44
As well as inflammation, myocardial injury occurs as a result of cardiomyocyte apoptosis.45 In fact apoptosis has been shown to represent the major independent form of myocyte cell death during, and immediately after infarction.46 Hypoxia induces apoptosis, this phenomenon having been demonstrated in many cell types.47 A study using rat myocardium has shown that apoptosis occurring during 45 minutes of ischaemia advances substantially during 3 hours' reperfusion. In an effort to elucidate the regulation of hypoxia-induced apoptosis in the myocardium, experiments have been undertaken to examine levels of certain pertinent proteins. For example, co-localisation of apoptotic cells occurs with the cysteine protease caspase 3, which belongs to a family of enzymes common in various models of mammalian programmed cell death.48 There is also evidence for a role for the enzyme c-Jun kinase, which is induced upon reoxygenation.49 The role of p53, which is a protein central to the regulation of tumour cell apoptosis, is dependent on other tumour microenvironmental factors such as pH.50 Apoptosis found in hypoxic cardiomyocytes has been found to be concomitant with raised p53 activity, indicating a critical role for p53 activated pathways.51 However, it has been demonstrated that hypoxia-induced apoptosis seen in acute myocardial infarction also occurs in the absence of p53.52
Diagnosis of acute myocardial infarction is facilitated by raised levels of the enzymes lactate dehydrogenase (LDH) and creatinine kinase (CK), levels of which increase in response to hypoxia/reoxygenation, along with a corresponding increase in levels of nitric oxide.53 It is logical that the vasodilatory effect of nitric oxide contributes to the inflammatory response, ultimately leading to reperfusion injury. The production of this chemical is catalysed by the enzyme nitric oxide synthase (NOS). There are three types. Perhaps the most pertinent when considering cardiac ischaemia is eNOS, which is produced constitutively by blood vessel endothelia, and is responsible for the synthesis of nitric oxide or, as it was previously known, endothelium-derived relaxing factor (EDRF). The two other forms are cytokine-inducible NOS (iNOS), which is calcium independent and important in the immunogenic response of T-lymphocytes, macrophages and reticulo-endothelial cells, and nNOS, which is the neuronal form of nitric oxide synthase.34
Although it is known that nitric oxide is released from cardiomyocytes in response to hypoxia/reoxygenation,53 a decreased production of endothelial nitric oxide by both acute and chronic hypoxia has been observed in pulmonary arteries.54 Likewise, experiments undertaken to elucidate the effect of acute or chronic ischaemia/hypoxia on the expression of these enzymes in the cardiopulmonary system has failed to provide an equivocal answer, providing evidence for both up- and down-regulation of these enzymes in hypoxic conditions.55–59 The diverse role of nitric oxide as a haemodynamic agent, acting both locally and systemically on a variety of tissues in response to different degrees of hypoxia, precludes a full understanding of the subject. However, further investigation of the nitric oxide pathways will uncover useful therapeutic targets. These include a role for nitric oxide stimulation in the protection of transplanted vascular organs from the effects of ischaemia/reperfusion,60 and the use of gene therapy where eNOS could be produced under hypoxic conditions to treat pulmonary arterial hypertension.61 There is also believed to be a link between the cardio-protective effects of ACE inhibitors, such as ramipril, and its stimulation of nitric oxide production.62
The role of hypoxia in solid tumours, cardiac ischaemia and the aetiology of inflammation and reperfusion injury has been discussed in detail, and therefore extends to conditions such as rheumatoid and other arthritides, chronic inflammatory bowel disease and skin conditions such as eczema and psoriasis. Inflammation and, therefore, hypoxia feature strongly in the mechanisms producing vasculopathies, such as vasculopathy following transplantation,63 diabetic inflammatory vasculopathy64 and diabetic neuropathy.65 Chronic respiratory diseases such as chronic bronchitis, cystic fibrosis and tuberculosis cause inflammation, and it is logical that the accompanying decrease in lung function leads to a hypoxemic state.
Reperfusion injury, aside from its impact on cardiac function, also leads to cerebral, hepatic, renal66 and intestinal complications such as hypoxia induced necrotising enterocolitis.67 Hypoxia induced oxyradical formation has been implicated in the evolution of atherosclerotic lesions,68 and decreased blood flow to the extremities, with the ensuing local hypoxia, can lead to ulceration.69 Meanwhile, in the area of sports medicine, repetitive activity resulting in a deficiency of nutrients and tissue hypoxia can precede tendon degeneration.70
Bioreductive drugs are currently being developed as a means of selectively targeting hypoxic tumour cells. Hypoxia directed drug delivery systems might offer the potential for selective treatment in a whole variety of conditions manifesting chronic hypoxia and inflammation, which are routinely treated using glucocorticoids and/or non-steroidal anti-inflammatory drugs and other therapeutic strategies. This offers the advantage of tissue specificity with reduced toxicity.
Bioreductive drugs were initially designed to diagnose the presence of, and be toxic towards, the hypoxic cells in tumours. There are four main classes of bioreductive drugs currently undergoing clinical or preclinical evaluation in the treatment and prognostication of solid tumours. These are:
For these agents to act as therapeutic or diagnostic agents in hypoxic conditions, a reductive activation must take place (Figure 5).71
Figure 5: The hypoxia-mediated activation of bioreductive drugs to give cytotoxic or diagnostic entities.
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The formation of the one-electron reduced intermediate is in equilibrium with its reoxidation by molecular oxygen, which results in "futile cycling" and the production of a superoxide radical, thought to be responsible for the aerobic toxicity seen in bioreductive cancer agents undergoing clinical trials. The position of this equilibrium depends upon the concentration of the drug, cellular oxygen tension and its redox potential. Consequently, prodrugs vary in the extent of hypoxia necessary for their bioreduction.72 Activation of these prodrugs is catalysed by various families of reductases, the most important to be considered being the cytochrome P450 reductase and cytochrome P450 family, which are the focus of much research into bioreductive drug metabolism.73,74 A scheme for the activation of the bioreductive drug tirapazamine is shown in Figure 6, which involves a hypoxia-dependent one-electron reduction to an intermediate tirapazamine radical. Hydrogen abstraction from DNA in the cell results in the formation of the 2-electron reduction product SR 4317, which is not toxic to normoxic or hypoxic cells. The intermediate tirapazamine radical, however, acts as the cytotoxic species, inducing the formation of single DNA strand breaks.72
Figure 6: The metabolic activation of tirapazamine induced by hypoxia. The metabolites SR4317 and SR4330 produced by a 2-electron reductive process are not toxic, whereas the intermediate tirapazamine radical is thought to act as a DNA strand breaker (taken from reference 75, with permission) |
Hypoxic cell populations are resistant to both radiotherapy and chemotherapy. The aim of bioreductive chemotherapy is not only to overcome this resistance, but also to exploit this feature by using agents that selectively kill these cells, rendering the tumour more responsive to other therapies, as well as reducing the chance of metastasis and relapse. In addition, it is hoped that another clinical benefit will be a reduced necessity for repeated treatment that may entail a poor response rate with an increased toxicity to the patient.
Clinical trials so far have investigated the role of bioreductive drugs in multimodality therapy, ie, in combination with radiotherapy or chemotherapy. Experimental systems have shown that hypoxia-selective drugs target hypoxic cell populations while either cytotoxic agents or radiotherapy target the surviving aerobic cell population. For example, the antitumour effectiveness of tirapazamine and RSU1069, a 2-nitroimidazole bioreductive drug, has been shown to be effective when used in combination with single high doses of X-rays. The radiation will preferentially kill aerobic cells, tumour response being dependent upon the extent of the remaining hypoxic cell population. The administration of a bioreductive drug immediately after irradiation reduces the number of hypoxic cells and improves tumour response.72 Fractionated radiotherapy in conjunction with bioreductive drugs is also effective, as it allows rehypoxiation, ie, reversion to hypoxic status between fractionations, which provides the conditions necessary for bioactivation of the drug.76
Bioreductive drugs have been shown to potentiate the response to other antitumour agents. Nitroimidazoles potentiate alkylating agents in a mechanism that involves modulation of thiol levels, inhibition of repair and alteration of alkylating pharmacokinetics, eg, misonidazole used with melphalan.76 Clinical trials of tirapazamine in combination with cisplatin are currently in phase III. The observed potentiation effect is schedule dependent, with maximum tumour cell kill seen when tirapazamine is administered approximately 2.5 hours before cisplatin.77 This potentiation appears to be related to the DNA repair capacity of a particular cell type. Evidence supporting this notion comes from a pair of ovarian cell lines, one of which is resistant to cisplatin (due to increased repair capacity). Killing of this latter line is not potentiated by tirapazamine, whereas the wild type cell line from which it is derived is 600 times more sensitive to the tirapazamine/cisplatin combination.78 Phase I clinical trials have defined the maximum tolerated dose of tirapazamine as 390mg/m2, alone and in combination with 75mg/m2 cisplatin. The combination shows no significant enhancement of systemic toxicity,77 the major side effects of tirapazamine being acute reversible hearing loss and muscle cramps.79 Phase I/II trials have shown this combination to be effective in malignant melanoma, metastatic and recurrent cervical, lung, bladder, and head and neck cancers. Phase III CATAPULT (cisplatin and tirapazamine against platinum alone in previously untreated lung tumours) trials conclude that tirapazamine in combination with cisplatin prolongs survival in patients with advanced non small cell lung cancer. One year survival is significantly improved and tumour response rate is doubled in comparison to cisplatin alone.80 Trials assessing the use of tirapazamine in combination with radiation, other agents such as vinorelbine, paclitaxel, fluorouracil, and various other two- and three-agent combinations are also taking place.
It is now acknowledged that the severity and advancement of disease, as well as the predicted response, should be considered when designing a treatment regimen for an individual patient. Tumour response to both bioreductive agents and radiation is modulated by the hypoxic fraction, ie, the proportion of hypoxic cells in a tumour. This varies spatially and temporally between individual tumours.
In order to individualise treatment with bioreductive drugs, it is desirable to obtain an accurate prediction of the hypoxic fraction of cells, or the extent of hypoxia, so that the drug may be rationally applied. Various methods of measurement, based on nitroimidazole hypoxia markers, have been investigated.81–83 However, any method used must account for cell populations that are transiently or acutely hypoxic, as well as those that are chronically hypoxic. Ideally, samples should be obtained at biopsy, or incorporate a non-invasive, non-toxic means of detection. This would be especially convenient as hypoxic fraction could be predicted from the same sample as other prognostic indicators, potentially reducing the number of necessary procedures. Methods that have achieved the greatest success so far include direct measurement of pO2, using a polarographic oxygen microelectrode, and bioreductive chemical markers.
Oxygen electrodes enable direct measurement of oxygen tension within tissue by means of a 300µm probe that contains a membranised gold microcathode 12µm in diameter. This is held at –700mV relative to a silver chloride cathode. To be an accurate predictor, the electrode must be capable of detecting the low oxygen tensions that are representative of radiobiological hypoxia. The spatial and temporal heterogeneity of pO2 values necessitates a rapid response time, this arrangement recording 90 per cent of available oxygen in 500ms.84 The value of oxygen electrodes has been demonstrated when measuring intra- and inter-tumour variability, though the extent of the variability also shows that a minimum of measurements are necessary to gain a true prediction of tumour oxygenation.4 Oxygen electrodes have been used successfully to measure tumour hypoxia in various sites such as cervix, breast, head and neck (for review see Stone85). However, the technique has obvious limitations, in that it is an invasive tool and necessitates a surgical procedure.
The use of bioreductive drugs as markers of hypoxia is possible due to the formation of intracellular adducts derived from the covalent binding of reduced metabolites to cellular components. These intracellular adducts can be imaged non-invasively using several methods, including PET (positron emission tomography),86 or by analysis of biopsy samples by flow cytometry and immunohistochemical techniques.82 The most prolific markers tested to date are the nitroimidazoles, particularly the 2-nitroimidazoles, some examples of which are shown in Figure 7. For example, NITP and pimonidazole, whose bound adducts are detected immunologically, whereas SR4554 can be successfully detected by fluorine-magnetic resonance spectroscopy. In contrast to their therapeutic counterparts, bioreductive markers can be used at much lower doses, which provides much optimism that these agents will be available for diagnosis not only of tumours but of non-malignant diseases as well.87
Figure 7: Examples of 2-nitroimidazoles used as bioreductive markers of hypoxia |
While the assessment of the level of hypoxia and/or the fraction of radiobiologically hypoxic cells in tumours is an important aspect for the rational application of bioreductive drugs, it will also be aided by a knowledge of the levels of cellular reductases which can activate these prodrugs under hypoxic conditions. As mentioned previously, one of the most important of these is P450 reductase. It has been shown that the cellular levels of this enzyme are important for the activation and hypoxic toxicity of tirapazamine, nitroheterocyclics and indolequinones.88,89 Thus it may be particularly advantageous to target these drugs towards tumours that are not only shown to be hypoxic but also have high levels of this reductase.71 Information on both of these requirements for the individualisation of bioreductive drug therapy may come from the use of the bioreductive drug "markers" of hypoxia detailed above, although quantitative relationships between the level of hypoxia, reductase expression and invasive and non-invasive measurement of the appropriate marker needs to be established.
Intrinsic markers of hypoxia offer the advantage of providing a non-invasive means of detection that can be carried out without the addition of exogenous agents at the time of biopsy. In oncology, the hypoxia-dependent product VEGF has been shown to exist in a variety of cancers.90 VEGF expression has been associated with areas of high vascular density (vascular hot spots)11 and has been identified as an adverse prognostic indicator. There is evidence to suggest that VEGF expression is related to the overall level of hypoxia in the tumours,91 but its relationship to pO2 measurements and the binding of the bioreductive hypoxia markers remains to be established. Other genes that could act as surrogates to predict the level of hypoxia might include platelet-derived endothelial cell growth factor (PD-ECGF) - which is a hypoxia-regulated enzyme - thymidine phosphorylase,92 and some glucose transporters, but as with VEGF their use still needs to be validated. Similarly, there is much current research directed towards the measurement of the levels of the transcription factor HIF-1 (hypoxia inducible factor 1). This heterodimeric protein regulates the expression of many hypoxia-regulated genes and may provide an even more precise measure of hypoxia in tumours and other diseased tissues.93
There is no doubt that hypoxia is present in solid tumours and is a feature of other debilitating diseases. This is providing a robust basis on which to develop novel diagnostic and therapeutic strategies for the treatment of these diseases. However, as we enter the new millennium, the call will be for greater individualisation of treatment. Pharmacists will have a central role in the identification of those patients most likely to respond to a given treatment, thus allowing intelligent clinical trial design and, later, dose adjustment and the avoidance of unnecessary treatment. The development of hypoxic markers, be it extrinsic or intrinsic, will help us meet this challenge.
ACKNOWLEDGMENTS Rachel Airley is supported by a CRISP (Collaborative Research Studentship Programme for Pharmacy) award from the Royal Pharmaceutical Society.
Rachel Airley and Jane Monaghan are graduate students and Ian Stratford is professor of pharmacy in the Experimental Oncology Group, School of Pharmacy and Pharmaceutical Sciences, University of Manchester, Oxford Road, Manchester M13 9PL. Correspondence to Miss Airley