ANTISENSE TREATMENT OF PULMONARY HYPERTENSION

Abstract: The invention herein described relates to a method to treat pulmonary hypertension by antisense therapy using ET-1 derived antisense molecules delivered to the lungs as a pulse/spike in an inhaler.

Description: ANTISENSE TREATMENT OF PULMONARY HYPERTENSION The invention relates to a method and means to treat, but not exclusively, pulmonary hypertension by functionally suppressing the product of the ET-1 gene which encodes a vasoconstrictor peptide the expression of which is correlated mainly with this disorder. Pulmonary Hypertension (PH) is a disorder in which the blood pressure in the pulmonary (lung) arteries is abnormally high in the absence of other diseases of the heart or lungs. PH is considered to be present when the mean pulmonary arterial pressure is greater than 25mmHg at rest or 30mmHg during activity. The normal mean pulmonary - artery pressure is approximately 14mmHg at rest. PH is classified according to whether the disorder is primary or secondary PH. Primary PH (PPH) is a relatively rare disorder affecting mainly women between the ages of 21 and 40 although men and children can suffer from the disease. The incidence annual rate is as high as 2 per million of the UK population. In the USA approximately 300 new cases of PPH are diagnosed each year. The exact cause of PPH is unknown, this is mainly due to the low incidence of PPH and the lack of an effective animal model in which to study the disease. However PPH is associated with cirrhosis, AIDS and can be familial. Secondary Pulmonary Hypertension (SPH) is present where there is an identifiable cause resulting in an increase in pulmonary arterial pressure. These include left venticular failure, chronic hypoxia, lung disease or alveolar hypoventilation and a number of systemic diseases such as lupus erythromatosis, systemic sclerosis, SPH may also result from chronic thrombo-anabolic disease. For both PPH and SPH the symptoms are similar. These include breathlessness, sycope (faints), angina (chest pain), headaches, fatigue, blurred vision, insomnia, and intestinal complaints. SPH is much more common and is a major killer in the Western world. There are currently a number of drug treatments that can help to ameliorate pulmonary hypertension. The successful clinical treatments include the continuous intravenous administration of prostacyclin (PGI 2 ) and inhaled nitric oxide (NO), for severe disease; for milder degrees of illness, anticoagulants and calcium channel blockers can be used to lessen symptoms and improve survival. In pulmonary hypertension, sufferers respond differently to different drugs therefore no single drug regime can be used to blanket treat sufferers. Moreover dosages have to be empirically decided due to the potential for adverse side effects. For example, systemic low blood pressure, nausea, angina, headaches and flushing. Therefore alternative strategies are required to provide therapeutic treatment of pulmonary hypertension. The product of the gene endothelin-1 (ET-1) has been shown to act as a potent vasoconstrictor and is involved in the remodelling of blood vessels of the lung to cause narrowing and obstruction. The product of the ET-1 gene is found in endothelial cells that line arteries and has been shown to be elevated in expression in individuals and in animal models that show pulmonary hypertension. Moreover, current successful treatments of clinical and experimental and hypertension using PGI 2 , and NO have been shown to inhibit the expression of ET-1. This correlative data is strongly suggestive that ET-1 is an important component in mediating pulmonary hypertension. Indeed, the drug BQ-123 has been shown to antagonise the activity of ET-1 receptors and relieve pulmonary hypertension resulting from congestive heart failure. In rat models, m NA to ET-1 was shown to decrease as a consequence of long term BQ-123 treatment. The problems of drug side effects, amongst other things, has lead to alternative therapeutic strategies to be devised. A considerable effort is being made toward developing gene therapy approaches to control disease pathogenesis and it is likely that in subsequent years this will provide a serious alternative to the use of pharmaceuticals. A recent strategy to interfere with the expression of a gene is that of antisense technology. In brief this strategy involves the use of a DNA or RNA molecule that is complementary to a region of a selected gene and is able to hybridise (bind) under physiological conditions to the targeted nucleic acid to prevent either transcription of the gene or translation of the mRNA encoded by the gene. The antisense molecule is often a short oligodeoxynucleotide (ODN). However the molecule may be an oligodeoxyribonucleotide, or a modified oligodeoxynucleotide, or a modified oligodeoxyribonucleotide; each of which are able to hybridised to a selected part of a gene, or mRNA, under physiological conditions. The modifications to oligodeoxynucleotides will be apparent to one skilled in the art. The exact region of the nucleotide sequence of the gene to which the antisense molecule is designed can be empirically determined. However it is common practice to design oligodeoxynucleotides to the 5' region of the gene (to interfere with transcription initiation) or the mRNA (to interfere with translation). As stated before, the region of the gene to which the antisense molecule is directed is determined by the efficiency with which the antisense molecule suppresses the gene of interest. Contrary to the above this may be the 3' region and is determined experimentally. The length of the ODN also has to be determined experimentally. Typically ODNs are 20- 30 nucleotides in length but may be much longer. Presently, a number of delivery methods are being explored for their effectiveness at delivering gene therapy to a specific tissue. Viral vectors and liposomes have been used with some success. Recently, cationic lipids chelated to a therapeutic DNA molecule have been shown to deliver the DNA molecule to the lungs (1). Cationic lipids are positively charged lipids which when combined with negatively charged DNA form a tight complex. Moreover, the lungs are coated in a surfactant, a major component of which is dipalmitoylphosphotidylcholine, which is cationic at physiological pH and which is internalised by endothelial cells that line the arterioles of the lungs. In our co-pending application (GB9805185.7 ref. 2) the delivery of an aerosol containing a vasodilator by a specialised inhaler is described. As an alternative to the continuous perfusion of the lungs using a nebuliser, the therapeutic composition, ie aerosols, is delivered intermittently and in short pulses using the so called "spike principle". By using this methodology the medicament is driven deep into the lung tissue only during inhalation. There are many lung diseases and conditions suffered by humans for which the preferred form of treatment involves delivering a medicament of the appropriate sort down a patient's airways into the lungs, where the medicament can act upon, and perhaps be absorbed into, the tissues of the lungs. The most effective treatments for these disorders involve the inhalation of some suitable chemical agent in inhaled air. The co-pending application describes the administration of nitric oxide, a known vasodilator, in the form of a pulse or "spike". In the treatment of the constriction of the small pulmonary arteries, the very short pulse of nitric oxide is provided at the start of the inhalation, such that the resultant bolus of nitric oxide mixture inhaled by the patient has a nitric oxide concentration high enough to have the desired therapeutic effect, even if admixed with some additional air, but is of such short duration (both in time and as a result, in physical amount) that, pushed by the following much larger volume of plain, and therefore nitric oxide - free, air/oxygen, it reaches deeper into the lungs, where it both acts on the small pulmonary arteries and is taken up into the capillaries, The invention therein described is a small (pocket-sized) hand held treatment apparatus that utilises the "spike" principle. The invention comprises a conventional nebuliser (having a reservoir in which is stored the medicament to be administered) driven by some suitable pressurised gas from a valved cylinder so as to deliver a medicament into a tube through which the patient is breathing (by mouth) normal air, the gas cylinder valve being controlled by a suitably programmed computing device that is fed data describing the pressure within the breathing tube and so is able to open and close the valve at, and for, a time such as to drive the nebuliser to deliver (to the tube and thence to the patient's lungs) a required pulse of medicament at any selected point within the patient's respiratory cycle. The idea of this is simply to ensure that a high concentration of the medicament reaches and affects the target area, and the target area alone, rather than having the whole of the lungs subjected to it. The apparatus of the invention incorporates a nebuliser in the reservoir of which is storable the medicament to be administered. The reservoir has a fine orifice through which its contents may exit and across which is blown a stream of gas along a pipe way. As the gas flows past the orifice it causes an external pressure drop (relative to the reservoir's internal pressure) resulting in some of the reservoir's contents being sucked out into the gas stream creating an aerosol-like cloud of fine drops or particles that is swept along the pipe way by the driving gas. The driving gas is contained within a valved cylinder, the operation of the valve being controlled by a computing means in response to the input from a pressure sensor. The pressure sensor is located within the breathing tube and provides the computing means with data about the air pressure there within, and thus about the patient's respiratory cycle. The invention therefore comprises a nebuliser with a reservoir containing a therapeutic composition which has contact with a gaseous supply through which the therapeutic composition is aerosolised. The delivery of the composition via the breathing tube is regulated by a valved cylinder which operates according to pressure data received from a pressure sensing means located in the breathing tube to determine the stage of the patient's respiratory cycle. The whole system is computer controlled to ensure that the pulse of therapeutic composition is delivered at the appropriate stage of the patient's respiratory cycle. We have used an inhaler employing the above 'spike' methodology and in one example the specialised inhaler described in GB9805185.7 to create an aerosol of ET-1 antisense molecules to perfuse lung tissue to relieve pulmonary hypertension. The designed antisense ET-1 molecules will target either the ET-1 gene and/or the ET-1 mRNA to suppress the ET-1 gene product from the endothelial cells that line the lung arterioles and thereby relieve pulmonary hypertension. It is therefore an object of the invention to design antisense ODNs to ET-1 for use in the alleviation of pulmonary hypertension. It is yet a further object of the invention to deliver antisense ODNs to the lungs to alleviate pulmonary hypertension. According to a first aspect of the invention there is provided a method to treat pulmonary hypertension comprising administering a therapeutic composition to an individual, wherein said composition comprises at least one aerosolised antisense ET-1 molecule, delivered to the lungs by an inhaler during inhalation by a patient. Ideally said antisense material is of mammalian origin, and most ideally of human origin. It will be apparent from an analysis of the prior art that the biological effects of ET-1 are mediated through the receptors ET-A and ET-B. Therefore although embodiments of the invention will be described that target ET-1 with antisense ET-1 molecules it will be apparent that targeting ET-A and/or ET-B may achieve the desired result of suppressing ET-1 activity although not directly. Therefore the following description assumes that one skilled in the art would appreciate that the antisense strategy could be applied to suppress ET-A and/or ET-B activity. According to a further aspect of the invention there is provided a method to treat pulmonary hypertension comprising delivering aerosolised antisense ET-1, or ET-A or ET-B molecules to the lungs by an inhaler as a pulse/spike during inhalation. According to yet a further aspect of the method there is provided a therapeutic composition for treating pulmonary hypertension comprising; the non-coding strand of the ET-1 gene, or fragment thereof, as represented in Figure 1. It will be apparent from Figure 1 that the sequence presented is the sense strand of the ET-1 gene. Antisense molecules would be designed to represent sequence complementary to this sequence and can be easily deduced from the sense sequence. In a preferred method of the invention said antisense ET-1 molecule is a oligodeoxynucleotide, a modified oligodeoyxnucleotide, an oligodeoxyribonucleotide or a modified oligodeoxyribonucleotide represented by the DNA sequence shown in Figure 1, that is the non-coding strand of ET-1. Reference herein to modifications is intended to include without limitation modifications apparent to those skilled in the art that improve stability (both as a stored composition and during in vivo use; improve permeability across biomembranes or facilitate aerosolisation). In a preferred method of the invention the antisense molecule is between 10- 100 nucleotides in length. In yet a further preferred method of the invention said antisense molecule is greater than 100 nucleotides in length. In yet still a further preferred method of the invention said oligonucleotide has a G + C content of at least 50% and more preferably still a G + C content of between 50% and 60%. More preferably still said oligonucleotides do not contain a consecutive run of more than four bases of any one kind. In yet still a further preferred embodiment of the invention said oligonucleotide has a predicted melting temperature of at least 55°, and more preferably still, a melting temperature of between 55° - 80° . In a preferred embodiment of the invention there is provided at least one human ET-1 antisense oligonucleotides as represented in Table 1. Table 1 HsETl 17 CAG CCC AAG TGC CCT TTA AC HsET132 CTC AAA GCG ATC CTT CAG CC HsET318 AGC TCA GCG CCT AAG ACT GC HsET438 TGG CAG AAG TAG ACA CAC TC HsET779 TGG TCT CCG ACC TGG TTT GT HsET868 ATG TGC TCG GTT GTG GGT CA In yet a further preferred embodiment of the invention there is provided at least one rat ET-1 antisense oligonucleotides as represented in Table 2. Table 2 RnET198 ACA GCA GAG AGA AGA TCA CG RnET294 TGC ACT TCC TTC TCA GCT CG RnET466 GGA TCG CTT AGA CCT AGA AG RnET718 CTT GAT GCT GTT GCT GAT GG RnET795 AGT CAA TGT GCT CGG TTG TG RnET1081 ACT GTG TCT CTG CTC TCC GA It will be evident to one skilled in the art, and from the above description, that a reliable animal model to test the efficacy of therapeutic treatments for pulmonary hypertension currently does not exist. It would therefore be desirable to develop an animal model system to test the efficacy of antisense ET-1 molecules. The sequences identified in table 2 represent preferred rat sequences. However it will be apparent to one skilled in the art that oligonucleotides derived from other species may also be useful in practising this method of the invention. Particularly those sequences that have a high degree of sequence homology to those sequences presented in Tables 1 and/or 2. According to a further aspect of the invention there is provided a method for determining the efficacy of antisense ET-1 molecules comprising exposing a known hypersensitive animal model system to antisense ET-1 for studying molecules and observing the effects of same on pulmonary hypertension. In a preferred embodiment of the invention said therapeutic treatment is based on antisense therapy. Ideally said antisense therapy is based on the ET-1 sequence and more preferably still those sequences represented in Table 1 and/or Table 2, and/or sequences homologous or analogous thereto, wherein said homology is at least 50%, ideally 75% and preferably at least 90%. In yet a further preferred method of the invention there is provided a modified antisense ET-1 molecule as herein described for the treatment of pulmonary hypertension. It will be apparent to one skilled in the art that a number of potential modifications can be made to the antisense oligodeoxynucleotide molecules to improve efficacy and/or stability of said molecules. These include modifications to the phosphodiester bonds between nucleotides, (eg the inclusion of peptide bonds to form peptide nucleic acids). Other modifications may be to the bases and/or sugars by the covalent attachment of chemical groups to specific sites in the sugar and/or base. All of these modifications will not affect the binding properties of the antisense molecule to its target site in ET-1. According to a further aspect of the method there is provided an antisense molecule adapted to hybridise to the transcriptional promoter region of the ET-1 gene to inhibit transcription of said gene. It will be apparent from an analysis of the prior art that the biological effects of ET-1 are mediated through the receptors ET-A and ET-B. Therefore although embodiments of the invention will be described that target ET-1 with antisense ET-1 molecules it will be apparent that targeting ET-A and/or ET-B may achieve the desired result of suppressing ET-1 activity although not directly. Therefore the following description assumes that one skilled in the art would appreciate that the antisense strategy could be applied to suppress ET-A and/or ET-B activity. It will be apparent to one skilled in the art that the antisense ET-1 molecule used in this way can be designed to either the sense strand or antisense strand of the ET-1 transcriptional promoter as the target sequence is double stranded genomic DNA. According to yet a further aspect of the invention there is provided an antisense molecule adapted to hybridise to splice junctions between exons and introns of prepromessenger RNA encoding the ET-1 protein. It will be apparent to one skilled in the art that the expression of the ET-1 gene can be inhibited by interfering with the splicing of introns from the pre mRNA of ET-1 to prevent the formation of a mature, translatable mRNA. According to a further aspect of the invention there is provided an inhaler comprising means for administering the aforedescribed antisense molecules encoding ET-1 nucleic acid wherein said administration is via a spike or pulse and preferably only during inhalation by the patient. According to yet a further aspect or embodiment of the invention said inhaler is disposable and adapted to receive a cartridge containing antisense molecules encoding the ET-1 nucleic acid composition. According to yet a further aspect of the invention there is provided a method for administering the composition of the invention comprising; i) adding or providing the antisense molecular composition in a suitable diluant, carrier or excipient and in dosage form to, or in a reservoir of, an inhaler; ii) aerosolising said composition by a suitable means; iii) detecting the air pressure within the inhaler by a suitable sensing device to determine a patient's respiratory cycle; and iv) delivering a prescribed dose of the afore described therapeutic composition to the patient's lungs during a selected inhalation phase of the patient's respiratory cycle. In yet a further aspect of the method there is provided a kit used to treat pulmonary hypertension comprising; a therapeutic antisense composition as hereindescribed; an inhaler, including a nebuliser, a reservoir for containment of said composition, an air pressure sensing device and a valve containing pressurised cylinder means to regulate the dosage of said composition. The invention hereindescribed therefore provides a means of controlling pulmonary hypertension using aerosolised antisense molecules to the ET-1 gene, and/or mRNA, to inhibit the vasoconstrictor activity of ..the ET-1 peptide. Moreover, the delivery of the antisense molecule makes use of an inhaler that functions during patient inhalation to drive the therapeutic composition deep into the lungs to deliver the antisense molecule to the endothelial cells that line the arterioles of the lungs and thereby target those blood vessels that result in pulmonary hypertension. An embodiment of the invention will now be described, by example only, and with reference to the following Figure and methods wherein; Figure 1 represents the genomic DNA sequence of human ET-1. Antisense Design Protocol Table 1 Human sequences HsETl 17 CAG CCC AAG TGC CCT TTA AC HsET132 CTC AAA GCG ATC CTT CAG CC HsET318 AGC TCA GCG CCT AAG ACT GC HsET438 TGG CAG AAG TAG ACA CAC TC HsET454 CCA AAT GAT GTC CAG GTG GC HsET779 TGG TCT CCG ACC TGG TTT GT HsET868 ATG TGC TCG GTT GTG GGT CA Table 2 Rat sequences RnET198 ACA GCA GAG AGA AGA TCA CG RnET294 TGC ACT TCC TTC TCA GCT CG RnET466 GGA TCG CTT AGA CCT AGA AG RnET718 CTT GAT GCT GTT GCT GAT GG RnET795 AGT CAA TGT GCT CGG TTG TG RnET1081 ACT GTG TCT CTG CTC TCC GA The above sequences were designed by identifying regions of loose predicted secondary structure and choosing sequences of 20 base pairs meeting the following criteria; a) 50-60% G+C content b) predicted melting temperature of 55-80 °C c) <4 runs of bases of any kind Regions of loose secondary structure were defined as sequences of >8 adjacent bases in the mRNA sequence for which there were no predicted intra-molecule Watson-Crick base pairing determined using the Wisconsin Genetics Computer Group software package RNAFOLD. The mRNA sequence was then imported into the 'primer' package on the Sheffield 'biocomp service' and complemented (G for C, T for A etc.). The regions of loose predicted secondary structure were sought and the oligo criteria were selected. The 3' most bases were aligned with the region of no predicted secondary structure. If the sequence highlighted met all set criteria the sequence and position was recorded. If the sequence failed to reach the criteria the highlighted 20 base sequence was shifted 5' until either a sequence was identified meeting the criteria or the <5 bases remained within the region of loose predicted secondary structure at which point the region was rejected. When more than one 20 base sequence met the criteria at a particular region of loose predicted secondary structure the sequence with the most central homology to that of the loose predicted secondary structure was selected. Current data relating to the efficacy of ET-1 antisense material is in vitro cell culture work. Selected antisense molecules are transfected into cells in culture and the level of ET-1 protein determined by western blotting and/or immunofluoresence using ET-1 antisera. A down regulation of ET-1 protein results when selected antisense molecules are used. Currently we are unable to identify the mechanism by which ET-1 protein is down regulated (ie destabilisation of mRNA, inhibition of translation). Further work will optimise conditions with respect to providing a suitable composition for use in in vivo studies. The working of the invention is undertaken by using conventional techniques which will therefore not be described herein in great detail. Suffice to say, that the hereindescribed antisense material is manufactured in conventional manner using laboratory techniques and then suspended in a suitable fluid for the purpose of delivery (3). When thus manufactured the antisense material is suitably contained, ideally in a cartridge, which cartridge is adapted for use in a conventional inhaler and more preferably the inhaler described herein (2). In this way, the invention is worked in a conventional fashion. 1 Delivery of genes using cationic lipid US patent no. 5641662 2 Inhalers. UK patent application no. 9805185.7. 3 Nyce, J.W. and Metzger, WJ. DNA antisense therapy for asthma in an animal model. Nature 285: p721-725. 1997.

Claims: CLAIMS 1. A method to treat pulmonary hypertension comprising administering a therapeutic composition to an individual, wherein said composition comprises at least one aerosolised antisense ET-1 molecule, delivered to the lungs by an inhaler during inhalation by a patient. 2. A method according to Claim 1 wherein said composition is delivered to said lungs as a pulse/spike during inhalation. 3. A therapeutic composition for treating pulmonary hypertension comprising a non-coding strand of the ET-1 gene, or fragment thereof, as represented in Figure 1; and, optionally, in combination with a suitable carrier. 4. A therapeutic composition according to Claim 3 wherein said non- coding strand of the ET-1 gene is a oligodeoxynucleotide, or a modified oligodeoxynucleotide, or a oligodeoxyribonucleotide, or a modified oligodeoxyribonucleotide. 5. A therapeutic composition according to Claim 3 or 4 wherein said non-coding strand of the ET-1 gene is between 10-100 nucleotides in length. 6. A therapeutic composition according to Claims 3 or 4 wherein said non-coding strand of the ET-1 gene is greater than 100 nucleotides in length. 7. A therapeutic composition according to Claims 3-6 wherein said oligonucleotide has a G + C content of at least 50%. 8. A therapeutic composition according to Claims 3-7 wherein said oligonucleotide has a G + C content of between 50% and 60%. 9. A therapeutic composition according to Claims 3-8 wherein said oligonucleotide does not contain a consecutive run of more than four bases of any single kind. 10. A therapeutic composition according to Claims 3-9 wherein said oligonucleotide has a predicted melting temperature of at least 55° C. 11. A therapeutic composition according to Claims 3-10 wherein said oligonucleotide has a predicted melting temperature of between 55° - 80°C. 12. A therapeutic composition according to Claims 3- 11 wherein said antisense ET-1 molecule is of human origin and comprises at least one from; i) CAG CCC AAGTGC CCTTTAAC ii) CTCAAA GCG ATC CTT CAG CC iii) AGCTCAGCG CCTAAGACT GC iv) TGG CAGAAG TAGACA CACTC v) TGGTCT CCGACCTGGTTT GT vi) ATGTGC TCG GTT GTG GGT CA or an oligonucleotide sequence homologous therewith. 13 A therapeutic composition according to Claims 3-12 wherein said antisense ET-1 oligonucleotide is of rat origin and comprises at least one from; i) ACA GCA GAGAGAAGATCA CG ii) TGC ACT TCC TTC TCA GCT CG iii) GGATCG CTTAGA CCTAGAAG iv) CTT GAT GCTGTTGCTGAT GG v) AGT CAATGT GCT CGGTTGTG vi) ACT GTGTCT CTG CTC TCC GA or an oligonucleotide sequence homologous therewith. 14. A therapeutic composition according to Claims 3- 11 wherein said non- coding strand of the ET-1 gene is adapted to hybridise to the transcriptional promoter region of the ET-1 gene to inhibit transcription of said gene. 15. A therapeutic composition according to Claims 3- 11 wherein said non-coding strand of the ET-1 gene is adapted to hybridise to splice junctions between exons and introns of prepromessenger RNA encoding the ET-1 protein. 16. A method for detemining the efficacy of a therapeutic composition in the treatment of pulmonary hypertension comprising administering a composition according to Claims 3-15 to a selected human or animal and observing the effects of said composition on pulmonary hypertension. 17. An inhaler comprising means for administering the therapeutic composition according to Claims 3-15, wherein said therapeutic composition is administered via a pulse or spike during use of the inhaler. 18. An inhaler according to Claim 17 wherein the inhaler is disposable. 19. An inhaler according to Claim 17 wherein the inhaler is adapted to receive a cartridge containing antisense molecules according to Claims 3- 15. 20. An inhaler according to Claims 17 -19 wherein said inhaler comprises a removable cartridge wherein said therapeutic composition is stored prior to use. 21 An inhaler according to Claims 17-20 wherein said inhaler comprises a rechargeable cartridge in which said therapeutic composition is stored prior to use. 22. A method for administering a therapeutic composition according to Claims 3-15 comprising; i) adding or providing said therapeutic composition in a suitable dilutant, carrier or excipient and in dosage form to, or in a reservoir of, an inhaler; ii) aerosolising said composition by suitable means; iii) detecting the air pressure within the inhaler by suitable sensing device to determine a patient's respiratory cycle; and iv) delivering a prescribed dose of the therapeutic composition to the patient's lungs during a selected inhalation phase of the patient's respiratory cycle. 22. A kit for treating pulmonary hypertension comprising a therapeutic composition according to Claims 3-15, an inhaler according to Claims 17-21, an air pressure sensing device, and a valve containing pressurised cylinder means to regulate the dosage of said composition.

Inventors: HIGENBOTTAM TIMOTHY, MCCORMACK KEITH, SMITH ADRIAN, HIGENBOTTAM, TIMOTHY, MCCORMACK, KEITH, SMITH, ADRIAN
Applicants: HIGENBOTTAM TIMOTHY, MCCORMACK KEITH, SMITH ADRIAN, UNIV SHEFFIELD, HIGENBOTTAM, TIMOTHY, MCCORMACK, KEITH, SMITH, ADRIAN, UNIVERSITY OF SHEFFIELD
Application year: 1998
Main classification: C12N15
IPC codes: A61K31, A61K38, A61M11, A61M15, A61P3, C12N15