The impact of molecular medicine upon early cardiovascular drug development

Introduction

Heart disease remains the leading cause of death and morbidity in the western world despite significant advances in cardiovascular (CVS) therapeutics. Since the 1960s clinical pharmacologists have been very much involved in bringing forward new classes of CVS drugs. This started with the introduction of β-adrenoceptor blockers in the early 1960s and has continued with calcium antagonists, ACE inhibitors and more recently endothelin antagonists. The principles for classical drug design have been based upon interaction with: receptor sites (β-adreno-ceptor blockers), enzymes (thrombolytics and anticoagulants) and ion channels (calcium antagonists, antiarrhythmics). The advent of molecular biology has added a fourth principle for CVS drug development – that of molecular therapeutics. While classical approaches will still provide new CVS drugs (e.g. platelet IIb/IIIa receptor antagonists and directly acting thrombin inhibitors), there is now anticipation that molecular therapeutics will provide a very specific means of intervention thus potentially avoiding some of the problems associated with the non specificity of conventional approaches, e.g. side-effects associated with lack of cardioselectivity with β-adrenoceptor blockers, risk of bleeding with anticoagulants, cough with ACE inhibitors, controversy following retrospective analysis of the use of calcium antagonists.

In the broader sense molecular therapeutics includes both gene therapy and oligonucleotide therapy. However the advances in genetic analysis (identification of polymorphisms associated with diseases) mean that there may be a further impact upon drug development – namely the use of known gene polymorphisms to select patient populations at risk. We will review the status of each of these and prospects for the future.

Genetic polymorphisms

Environmental factors such as diet and smoking play an important part in determining the susceptibility to coronary artery disease (CAD). However, these are not the only predictors of risk – genetic predisposition also plays a role. Unlike the long QT syndrome or hypertrophic cardiomyopathy which are monogenic disorders, CAD represents an interplay between the environment and genetic factors. Thus identifying the genetic factors and using this information will be a key challenge for drug development in CAD. Below are summarized some gene polymorphisms identified within several aspects of cardiovascular disease – thrombosis, hypertension and hyperlipidaemia.

Thrombosis

One of the first important polymorphisms which could affect CVS drug development to be discovered was the insertion/deletion polymorphism for the angiotensin converting enzyme (ACE), this gives rise to the II, ID and DD genotypes. ACE has a significant impact on the cardiovascular system, the levels are partially under genetic control. The DD genotype is associated with higher levels of angiotensin I and has been associated with increased risk of myocardial infarction [1], although this has been questioned [2]. The full ramifications of the ACE gene will be reviewed in a subsequent paper in this series. There is evidence that patients with the D polymorphism respond differently to ACE inhibitors. A poorer response has been seen in terms of effect in diabetic nephropathy, the DD polymorphism showed a steeper decline in renal function [3]. A recent small study in 34 heart failure patients examined response with respect to ACE genotype [4]. The II genotype showed a greater hypotensive response to captopril and a less favourable response in terms of change in renal function. Preliminary data for angiotensin II receptor blockade in hypertensives, indicate a larger diastolic blood pressure response in patients with the II genotype [5].

A polymorphism for the platelet glycoprotein IIb/IIIa receptor – P1^A2 has been identified and claimed to correlate with increased risk of coronary thrombosis and stenosis [6, 7], but this has also been disputed [8]. Identification of a polymorphism related to a receptor site allows speculation about the implications for development of drugs acting at the fibrinogen receptor site (the overall role for this class of drugs is currently the subject of much debate and interest), in addition to identifying patients at increased risk of CVS disease.

Another polymorphism of potential importance in thrombosis is that of plasminogen activator inhibitor 1 (PAI-1) which inhibits the conversion of plasminogen to plasmin and therefore fibrinolysis. Increased levels of PAI-1 are thus a risk factor for thrombosis and an I/D polymorphism in the promoter region of the PAI-1 gene is associated with levels of PAI-1; the 4G allele is associated with higher levels of PAI-1 and is cited a factor for MI [9]. Again this association has been found to be inconsistent [10]. The HindIII polymorphism for PAI-1 has been shown to correlate with the degree of coronary artery disease, but again in a small study [11].

There are also emerging relevant polymorphisms for the coagulation system that will need to be taken into account. For example a change in the gene encoding for Factor V (so called Leiden mutation, which substitutes arginine for glutamine at position 506), confers resistance to inactivation by activated protein C – this occurs in 2–15% of Caucasians [12]. The Factor V Leiden mutation is the most common genetic risk factor for venous thrombosis, heterozygotes having a seven fold increased risk [13].

Several polymorphisms for Factor VII have been found to be associated with either increased or decreased risk of MI [14]. A transition of G to A at nucleotide 20210 in the prothrombin gene is associated with higher prothrombin levels, such subjects being at increased risk of having a venous thrombosis independently of Factor V Leiden. One case control study found an odds ratio of 3.1for this polymorphism [15], similar to that of 3.7 found by Leroyer et al.[16]. The aforementioned ACE DD genotype has also been shown to be a risk factor for the development of venous thrombosis following hip replacement. The homozygous DD genotypes having an 11-fold and the heterozygous ID genotype a 5 fold increase in risk [17].

It seems evident that identification of several concomitant polymorphisms will yield a population, which is at significant increased risk as well as selecting patients who may respond differently to antithrombotic drugs. A recent study in pregnancy showed that when testing for mutations in Factor V, prothrombin and methylenetetrahydrofolate reductase, over 50% of women with obstetric complications had a thrombophilic mutation by comparison with an incidence of under 20% in those with normal pregnancies [18]. The benefits of multilocus genotyping have been highlighted for cardiovascular disease using a prototype assay to test for 35 sites within 15 genes [19]. The approach is now being automated with the advent of DNA arrays or ‘gene chips’[20]. Up to several hundred thousand oligonucleotide or cDNA sequences are gridded onto glass or polymer matrix. Application of the target gene to the microarray under defined hybridization conditions of different stringency will facilitate the rapid screening for single nucleotide polymorphisms and mutations with unprecedented speed.

The advent of DNA arrays carries significant implications for drug development. This will be especially true in early phases, where there is increasing pressure to select in the most efficient manner those candidates that show greatest potential and similarly to stop unnecessary development of those that do not. Therefore, in future patients may be selected on the basis of genetic screening – either because they have an increased risk for the indication in question and so smaller numbers can be used in assessing ‘efficacy’ in early studies or alternatively genetic analysis may indicate that certain patients will respond differently.

One note of caution is that, as noted above, there have been instances of relatively small studies showing an association according to a given polymorphism, only to be refuted by larger studies.

Hypertension

Towards the end of the last century various unifying theories were proposed concerning the pathophysiology of essential hypertension (defects in sodium transport, increased sympathetic tone), however, these did not seem to be consistent with experience that only a minority of patients are controlled adequately by drug therapy, i.e. essential hypertension should not be considered as one disease. Thus it cannot be expected that one single antihypertensive drug can be effective in all patients. More recently the genetic bases surrounding Mendelian forms of hypertension have been identified in relation to: defects in steroid metabolism and ion channels plus overproduction of catecholamines. Essential hypertension represents a more complex interplay between genetic and environmental interactions, but the genetic information gained from Mendelian causes should be of some relevance to essential hypertension [21, 22].

The complexities of the genetics of hypertension will be uncovered in a separate review in this series. As further information is gained about the genetic determinants of hypertension it will be important that patients undergo careful characterization of both phenotype and genotype. For example there are various other conditions which cosegregate with hypertension, e.g. insulin resistance, dyslipidaemia. In future management of essential hypertension could be based upon guidance from genetic analysis in addition to phenotypic observations, e.g. effect of dietary salt.

Uncovering the genetics of hypertension will undoubtedly give further impetus for new approaches to the management of hypertension. Genetics may play a role in individualizing hypertensive patients' treatment [23] and determining how aggressively to treat individual hypertensives [24].

Dyslipidaemia

If hypertension represents a situation meriting careful dissection of environment and genetics, this would appear to be even more so in the case of lipid polymorphisms. Higher lipid levels are associated with the genetic variants for apoB and apoE and LPL [25]; a recent study has shown a higher frequency of the M1 allele of the apoA1 gene in relatively young males with unstable angina [26]. Such associations can be affected by environmental factors such as smoking and alcohol intake. In patients with established coronary artery disease deciding the contribution of a lipid polymorphism to the risk of a patient having a future cardiovascular event remains to be determined, but, a clearer role can be anticipated in primary prevention, to determine which subjects will be in need of early intervention before manifestation of the phenotype.

The use of a lipid polymorphism has been illustrated recently by the discovery that a variation at the gene locus of the cholesteryl ester transport protein (CETP) is associated with the progression of coronary artery disease [27]. The presence of this variant was associated with higher plasma CETP concentrations and lower HDL concentrations. Intriguingly patients with the B1 variant responded to pravastatin whereas patients without the variant showed no decrease in the progression of atherosclerosis when taking pravastatin.

Thus it appears possible to custom select for a clinical trial, a patient class in whom a response to lipid lowering treatment is the desired outcome. In view of the recent findings it should be possible to target the right drug to the right patient. However, this may represent the opening of a ‘Pandora's box’. Although, increasing the certainty about the response in a given patient group when selecting by genotype, this approach means a smaller target population, with attendant commercial implications for the size of the market for the drug in question. Also for those patients not fitting the genotype, other suitable means of treatment will need to be identified.

Oligonucleotide therapy

Antisense oligonucleotides are essentially short sequences (15–25 bases long) of modified DNA. Their absolute mechanism of action has not been established, but they act to prevent translation of RNA, either by promoting its attack by RNase or by sterically interfering with transcription of mRNA. Thus in principle once a target sequence of DNA has been identified, an oligonucleotide can be constructed and the gene expression switched off. In reality turning this into practical therapeutics has proved to be significantly more difficult. Firstly, initial oligonucleotide sequences were not stable as they were degraded by exonucleases – this has required the modification of the sugar backbone, e.g. phosphorothioate. Further improvement here is probably required as phosphorothioates are associated with side-effects such as thrombocytopoenia and increased transaminases; second generation mixed backbone oligonucleotides offer the potential for lower side-effects [28]. Secondly, as administration is easier on an acute basis, this limits the range of indications which could be treated by this approach. Thirdly, there has been debate over the specificity of the antisense effect – it is possible that effects may be mediated by the oligonucleotide binding to certain proteins – aptameric effects [29], although aptamers are now seen as an opportunity.

There was hope that antisense therapy could reduce the incidence of restenosis following balloon angioplasty, especially in view of the number and range of conventional pharmacological approaches which have failed. Several animal studies have indicated that this approach is feasible and targets used have been the protooncogenes c-myb and c-myc, as well as combinations directed against cell cycle regulatory genes.

The first clinical trial of antisense in angioplasty was undertaken with antisense to c-myc, but using patients who had undergone stenting. A relatively small trial (ITALICS) did not show any effect on in-stent volume obstruction as measured by intravascular ultrasound or upon luminal diameter as measured by coronary angiography (Inex Pharmaceuticals, personal communication). It is uncertain whether the stented population was the best group in which to test a single application of antisense as the stent can be viewed as an ongoing stimulus within the vessel wall. Use during angioplasty also highlights another problem – that of delivering an agent to the desired site. Many special catheters have been developed for delivery of agents into the vessel wall, initially starting with modifications of an angioplasty balloon using pressure, but progressing to the use of microneedles and iontophoresis [30–32]. If antisense is to be successful in the future, either local transcatheter drug delivery will need to be carefully optimized in tandem with work in the pig model of restenosis or a stent within the vessel may be used as a platform from which to deliver the agent.

Recently antisense to Bcl-x (an antiapoptotic mediator) has been used in a rabbit carotid injury model to demonstrate that Bcl-x is necessary for promoting lesion formation within the intima [33]. Antisense treatment resulted in apoptosis as confirmed by TUNEL (TdT-mediated dUTP nick end-labelling) staining and DNA laddering, with a consequent reduction of intimal lesions. This highlights that promoting apoptosis could be a novel therapy for dealing with both restenosis and atherosclerosis. Another aspect for antisense oligonucleotides illustrated that in addition to providing a therapeutic modality for certain diseases, they can be used to gain insight into disease mechanisms. An example of this approach is investigation of the function of protein kinase C isoforms [34].

Administration of antisense to the angiotensinogen II type I receptor has been shown to reduce blood pressure in several animal models [35, 36]. However, oral therapy would need to be developed for practical use in man.

Thus antisense has not yielded an immediate therapy for cardiovascular disease, but progress has been made in other areas. These include antisense molecules to PKC-α, c-raf kinase and Ha-ras for cancer being undertaken by ISIS Pharmaceuticals/Novartis and antisense against ICAM-1 for Crohn's disease by ISIS/Boehringer Ingelheim. Of particular note is that ISIS in collaboration with CIBA Vision have received regulatory approval to evaluate the antisense molecule, vitravene (inhibitor of CMV replication), in AIDS patients suffering from CMV retinitis.

For cardiovascular disease, related strategies to antisense are being pursued. The Dzau group has progressed decoy oligonucleotides. In contrast to the antisense approach, decoys are double stranded oligonucleotides, which act by inhibiting the binding of transcription factors to specific regions necessary to initiate gene expression. A decoy to the transcription factor E2F has been shown to prevent accelerated atherosclerosis in vein grafts. Inhibiting the binding of E2F prevents gene expression of cell cycle regulatory proteins, thus inhibiting smooth muscle cell proliferation. This approach has the advantage that the oligonucleotide can be added ex vivo to the graft, thus obviating concerns over local delivery. Initial results from use of E2F decoy in man – patients undergoing peripheral arterial bypass grafting – show that this approach is safe, results in delivery into the vein graft and produced a reduction of proliferating-cell nuclear antigen and c-myc expression [37].

The decoy approach may be extended in man to prevention of damage from myocardial reperfusion using a decoy to NF-κB. This transcription factor has a key role in expression of cytokines and adhesion molecules. Delivery of NF-κB decoy into rat coronary arteries prior to coronary artery ligation reduced damage following reperfusion [38]. For use in patients a myocardial specific delivery system will need to be developed and use confined to acute intervention in view of the physiological role of NF-κB [39].

Apatmers

Aptamers are therapeutic oligonucleotides that bind specific ligands. The sequence of the oligonucleotide determines the molecular structure of the aptamer and hence the specificity of the molecule that it binds to. Aptamers are derived by systematic evolution of ligands by exponential enrichment (SELEX) and can generate vast oligonucleotide combinatorial libraries [40]. The basic principle is to generate a starting library of sequences of between 10¹⁴ and 10¹⁵, although in a single synthesis it is possible to generate 10²⁰ or more different sequences. Having constructed the library, the aim is to challenge a particular target with the library of aptamers and to recover and systematically enrich oligonucleotides that specifically bind to the target molecule and may modify its function. Apatmers have proved applicable in the inhibition of thrombin [41] and vascular endothelial derived growth factor (VEGF) [42].

Gene therapy

Casting an eye back a decade on the state of CVS therapy, it is astounding to see the progress that has been made from bench to clinic to enable gene therapy in man. Gene therapy has already been used in man for vascular disease – although the first case was for a non-CVS indication – adenosine deaminase therapy. It is easy to conceive that gene therapy could be used for single gene disorders and also in cases where acute administration will provide an adequate means of administration. With regard to the former, the first human testing of gene therapy of the LDL receptor has been accomplished in patients with familial hypercholesterolaemia [43].

As an acute approach restenosis seems an appropriate target – the ability to intervene acutely at the time of angioplasty but to have a downstream effect in preventing restenosis occurring 3–6 months later. Examples of suitable genes that have been identified have been the herpes simplex thymidine kinase (HSV-TK) and retinoblastoma genes [44]. HSV-TK (so called suicide gene) is to be given in combination with the nucleoside analogue ganciclovir, which requires phosphorylation for incorporation into DNA and subsequent DNA termination. The retinoblastoma protein prevents cell cycle progression.

Hypertension is a polygenic disorder so it is unlikely that an approach based on a single gene could have widespread effects, although one could speculate that targeting sodium transport would seem to have particular relevance. For hypertension there is the added complication that it is a chronic disorder and as such it presents a challenge in designing a gene delivery strategy to ensure prolonged expression. Nevertheless there is a clinical need for a therapy which provides a long lasting effect. Candidate genes for antihypertensive therapy which have been identified include nitric oxide synthase and atrial natriuretic peptide [45].

Therapeutic angiogenesis is a strategy that can be successful when given acutely. We will first review the potential approaches and then discuss some of the development issues in assessing such gene therapy.

Possibilities for therapeutic angiogenesis

Preliminary results are now available for vascular endothelial derived growth factor (VEGF). Initially Isner and colleagues administered naked DNA for VEGF 165 by means of a hydrogel balloon catheter [46] and subsequently by intramuscular injection to patients with critical limb ischaemia (CLI) [47]. Effects were seen via the formation of new vessels by digital subtraction and magnetic resonance angiography. Evidence for clinical improvement (ulcer healing) has been reported in a small, uncontrolled study in Buerger's Disease (a severe form of peripheral arterial occlusive disease) [48]. Isner has now extended his work to give naked DNA VEGF to patients with severe angina by direct myocardial injection. An improvement in symptoms and myocardial perfusion was seen [49].

Naked DNA has the disadvantage of relatively poor uptake and duration of effect. In order to improve upon this, use is being made of viral vectors [50, 51]. Options available include: retroviral, adenoviral and adenoassociated vectors. The first only infect dividing cells, whereas adenoviral vectors can infect both dividing and nondividing cells, but have the potential to cause immune activation [52]. There is potential concern over readministration. Adenoassociated vectors are single stranded units of DNA and seem to offer promise in terms of transfection efficiency and lack of immune side-effects.

Crystal and coworkers have now administered VEGF isoform 121 to patients with CAD, using an adenoviral vector [53]. A further approach is via the use of fibroblast growth factor (FGF). Thus different isoforms of VEGF and other growth factors are now in clinical trials and there is a further approach to stimulating new blood vessel growth is via the use of transcription factors.

In addition to improving uptake by the use of vectors, it may be possible to control tissue gene expression by means of endogenous factors [54]. In the case of CVS gene therapy ischaemia can be used to control expression by means of hypoxia induced factors (HIFs). HIF-1 [55] activates transcription of hypoxia inducible genes by binding to a hypoxic response element (HRE) in the gene promoter. Genes that are regulated by HIF-1 include VEGF, erythropoietin, heme oxygenase-I, inducible nitric oxide synthase, and the glycolytic enzymes aldolase A, enolase I, lactate dehydrogenase A, phosphofructokinase I and phosphoglycerate kinase I. HIF-1 will activate the expression of all the VEGF isoforms and in theory, should elevate the levels of endogenous VEGFs to a higher constitutive level than achieved under hypoxia, given that HIF-1 has been shown to superactivate an HRE containing promoter under hypoxic conditions [56]. Results are awaited from in vivo preclinical studies. Recently, the John's Hopkins University granted Genzyme an option on exclusive worldwide rights to intellectual property and patents pertaining to HIF-1 for the treatment of occlusive vascular disease. Assuming that clinical trials now procede, this will set a precedent for use of a transcription factor in man. Transcription factor binding sites can be upregulated by shear stress [57, 58]. This has given rise to the possibility of a novel ‘gene switch’ approach, i.e. the use of inducible promoters within the vessel wall which contain transcription factor binding sites responsive to shear stress [59].

Development issues

Peripheral oedema has been seen following VEGF administration in CLI patients. The use of adenoviral vectors may also be associated with an acute inflammatory response [60], vasomotor dysfunction [61] and generation of thrombus [62]. Thus dose ranging studies in CLI/CAD will require careful assessment and monitoring for such effects as the dose is escalated.

Assessing effects in peripheral limbs has shown that vessels of 200–800 μm in diameter were formed using digital subtraction angiography, but current technology cannot assess new vessel growth below 200 μm. In the heart functional imaging (SPECT) coupled with stress testing has been used to provide an indicator of therapeutic success. This is essential prior to the setting up of larger clinical studies powered to examine the effect upon morbidity and mortality.

The initial work with VEGF in the heart has been accomplished by direct myocardial injection via a left thoracotomy. In the future a more direct and safer approach would be via a local delivery catheter. Such devices are now being developed to permit delivery either via iontophoresis (current assisted delivery) within the coronary artery or possibly via endomyocardial administration. Alternatively VEGF may be administered via minimally invasive coronary artery bypass grafting [63, 64]. A further possibility is use in combination with transmyocardial laser revascularization [65].

The above illustrates that many pharmaceutical company/biotechnology company/academic alliances are now taking forward angiogenic growth factors or employing the use of transcription factors. There will need to be critical assessment of dose response in early clinical trials. In addition to information on safety, evidence of efficacy and delivery will be required in order that the different approaches may be rated.

Delivery efficiency will need to be assessed for each mode of administration – change of vector, change of surgical procedure or use of special catheter. This may mean measurement of serum growth factor levels, measurement of growth factor mRNA or possibly biopsy to monitor for gene expression. Just as clinical pharmacologists and pharmacokineticists in the past have evaluated bioequivalence in changing from an immediate release oral formulation to a modified release, new skills will need to be mastered to evaluate the effect of a change in gene sequence or in the delivery vector. Assessing delivery will call for new pharmacokinetic skills. What should be measured for characterization of pharmacokinetics following gene delivery – levels of the gene or the expressed protein? Careful consideration is needed here [66]. For VEGF delivered to the heart, can we detect levels in the peripheral blood? This may be simply a measure of safety (by absence of levels), or is it feasible to measure levels local to the site of delivery as a measure of potential efficacy.

Delivery into the vessel wall (if this is the desired site) is not straight forward. It is complicated by high flow within the coronary circulation and also possible difficulty reaching the correct site in the vessel – the internal elastic lamina is an effective barrier. Extrapolation from animal models to man may not be easy, since there will be a more complicated pathology in the diseased vessel; the presence of atherosclerosis may even make delivery easier [67]. However, it seems prudent to first use isolated arteries to identify factors that may affect delivery [68].

From the above it is clear that those responsible for clinical development of gene therapy will need to work even closer with clinical pharmacologists in order to determine the best way to investigate effects in man.

Finally

Beyond angiogenesis it is not so hard to see that heart failure may benefit from gene therapy [69]. This indication still carries a high morbidity and mortality, so a radical approach is likely to be needed to have a further significant impact upon current management. Thus it may be possible to initiate the growth of new myocytes or switch off cytokine production. Indeed a special conference organized by the clinical section of the British Pharmacological Society recognized the need for further improvement in the management of heart failure and that gene therapy may play a role [70].

It has been recognized that apoptosis contributes to the underlying pathology of a number of cardiovascular disorders including heart failure [71]. In contrast to the accidental cell death of ischaemia, apoptosis refers to cell death involving caspases and suicide ligands. Pathologically it is characterized by DNA fragmentation – DNA run on agarose gels shows a laddering pattern or gel electrophoresis shows comet tails. Alternatively apoptosis can be assayed histochemically by TUNEL staining to detect free 3′ DNA ends. Thus if one can reverse/slow progression heart failure, it may be possible to use a molecular marker as a surrogate. TUNEL staining would require biopsy, but an alternative is to measure the soluble Fas [72]. Assessing apoptosis has been employed in a rabbit model of coronary ischaemia to demonstrate a protective effect of carvedilol, when it was shown to reduce apoptosis in the ischaemic area [73].

Conclusions

Molecular medicine in its broadest sense is going to have a significant impact upon the development of new cardiovascular drugs. Clinical pharmacological studies in man will be essential to permit an early evaluation of safety and efficacy.

The technology now exists to screen patients for multiple polymorphisms and on the basis of this it may be feasible to select a sub group who would be expected to respond more favourably to a drug – either because they are at increased risk or due to different response characteristics. Selecting the polymorphism and study design will require careful consideration in view of the tendency for conflicting results that have been seen to date when findings from relatively small studies have subsequently been refuted by larger trials.

Antisense therapy is probably not going to have an immediate impact for CVS disease; one way forward may be via optimization of local drug delivery. However, decoy oligonucleotides are now in clinical trials and offer hope for the management of occlusive vascular disease. One previously mooted downside for oligonucleotide therapy, aptameric effects, may now be exploited favourably in promoting angiogenesis.

Gene therapy to promote angiogenesis is now making progress and is being tested in various forms in clinical trials. Future success with therapeutic angiogenesis is likely to depend upon optimization of vector and mode of delivery. To assess this clinical pharmacologists are going to have to pay careful attention to assessment of safety, pharmacodynamic effects and pharmacokinetics. Success will depend upon preclinical and clinical scientists working together to rationalize delivery parameters in animal models and to follow up in man in order that such potential therapy can be optimized.