Additional therapeutic use

Details Additional therapeutic use Additional therapeutic use

1999-12-21

2002-10-15

Criares, Theodore J. (Department: 1614)

Drug, bio-affecting and body treating compositions

Designated organic active ingredient containing

Having -c-, wherein x is chalcogen, bonded directly to...

Reexamination Certificate

active

06465502

ABSTRACT:

Angiotensinogen, an &agr;2-macroglycoprotein, is cleaved by the enzyme renin into the decapeptide angiotensin I, which is itself only very slightly active biologically. In the next step of the cascade, two further amino acids are cleaved off by the action of the enzyme angiotensin converting enzyme (ACE), which is mainly bound in the endothelium, with the formation of angiotensin II. The latter is regarded as being one of the most powerful natural vasoconstrictors.
Angiotensin II interacts with specific receptors on the surface of the target cell. Success has by now been achieved in identifying receptor subtypes which are, for example, designated AT
1
receptors and AT
2
receptors. Studies on the renin-angiotensin system, particularly in relation to hypertension, have increased almost exponentially over the last decade. As a result, the number of receptors for Angiotensin II have now been identified and some of them have been cloned and analysed. Recently, considerable efforts have been made to identify the substances which bind to the AT
1
receptor, with active compounds of this nature frequently being termed angiotensin II antagonists. As a consequence of the inhibition of the AT
1
receptor, these antagonists can, for example, be employed as antihypertensives or for treating congestive heart failure.
The AT
1
and AT
2
receptors have also been studied for their distribution and biological properties and have been shown, despite a 30% homology, to have a very different distribution and activity.
The AT
1
receptor, which plays a major part in blood pressure regulation, has been found in the adrenal cortex, kidney, uterus etc. At a cellular level it has been found on fibroblasts, macrophages and smooth muscle cells (SMC).
In contrast, the AT
2
receptor has been found mainly in foetal tissues but also in adult especially in pathological tissue such as in ischaemic heart disease. Here it has been located on fibroblasts and endothelial cells.
The aim of the studies described hereafter is to evaluate the distribution of AT
1
and AT
2
receptors in the human lung using essentially the immunocytochemical and the in situ hybridisation methodologies.
Previously, specific antibodies have been made against epitopes of the AT
1
receptor but no such specific tools existed for the AT
2
receptor. Histological studies therefore depended on radio-labelled receptor antagonists together with autoradiography which only gave a relatively crude tissue localisation. In the last two years, however, specific well-characterised antibodies to the human receptor have become available and have therefore been used in the studies relied upon hereafter.
Methods and Materials
1. Antibody and in situ hybridisation (ISH) probe specificity and titration
In order to confirm the specificity of the immunocytochemical (ICC) and ISH studies, paraffin embedded blocks of normal human adrenal (cortex and medulla) are obtained from the archives of the Pathology Dept., University Hospital, Ghent, Belgium, and used as test material. AT
1
receptors are known to be predominantly located in the adrenal cortex and AT
2
in the medulla.
For the human lung studies, control material is obtained from autopsies where the patients have died from causes other than lung disease e.g. fatal accidents. Some of this material comes from Ghent as above, some from the archives of the Pathology Dept. of The Pennsylvania Hospital, Philadelphia, USA. Tissue containing small airways is selected as they appear to be more sensitive to injury.
All samples have been fixed in 10% buffered formalin as rapidly as possible post-mortem, dehydrated and embedded in paraffin wax. 3-5 &mgr;m sections are cut and mounted on silane coated glass slides.
In order to minimise any variations in the processsing, pairs of sequential sections are mounted on each slide, one for AT
1
and the other for AT
2
antibody exposure. Up to 20 slides are treated at the same time so that, with the exception of the primary antibody, al reagents including the chromogen are identical. Section thickness is therefore the only variable which could not be totally controlled.
2. Antibodies
Two AT
1
receptor antibodies, from Santa Cruz Inc., San Diego, Calf., USA, (clones N 10 and 306) are tested and found to give identical adrenal cortex receptor distribution.
The major part of the study is done with an AT
2
receptor antibody available from Santa Cruz (clone C18) which is tested and is found that it gives the identical staining pattern in adrenal medulla and lung as the first one.
All the antibodies have been rigorously tested by the commercial and individual suppliers for specificity and cross-reactivity.
3. ISH probes
PCR (Polymerase Chain Reaction) products were prepared and used as follows:
Oligonucleotides specific to human angiotensin II receptor type I (GenBank accession number M93394) and angiotensin II receptor type II (GenBank accession number U15592) were designed using the Oligo 5.0 software programme to homologous regions from both sequences (Table 1). cDNA from human bone tissue was prepared following standard methods (Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning: a Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). For PCR the following oligonucleotide primer pairs were used: angiotensin II receptor type I, 5′-CTggCTgACTTATgCTTTTTACTgACT-3′ (SEQ ID NO:1) and 5′-gATgCAggTgACTTTggCTACA-3′ (SEQ ID NO:2); (PCR product size 236 base pairs) and for angiotensin II receptor type II, 5′-ATTTACTCCTTTTggCTACTCTTCCTC-3′ (SEQ ID NO:3) and 5′-ggTCACgggTTATCCTgTTCTTC-3′ (SEQ ID NO:4) (PCR product size 489 base pairs). PCR amplifications were performed with 10 ng of template cDNA using a MJ Research PCR Cycle machine and the following PCR cycles: 1) 94° C./2 min, 2) 94° C./10 sec, 60° C./30 sec, 72° C./15 sec for 35 cycles, using High Fidelity Taq polymerase (Boehringer Mannheim) with components provided in the manufacturer's kit. Products of the PCR amplifications were identified by electrophoresis through a 0.8% agarose/TBE gel (Sambrook J, Fritsch E F, Maniatis T. Molecular Cloning: a Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989). To confirm the identity of the PCR amplification products, the DNA was eluted from the gel and cloned into the A/T cloning vector pMOSBlue (Amersham). Colonies containing a DNA insert of the correct size (Table 1) were fully sequenced on both strands to confirm their identity.
Each probe, both sense and anti-sense for the AT
1
and anti-sense only as described above, are labelled with fluorescin (FITC) and the presence of mRNA in the cells detected following hybridisation with the probe and use of a mouse anti-FITC probe plus the alkaline phosphatase anti-alkaline phosphatase (APMP) detection system. This labelling technique enhanced the detection of very low copy numbers.
4. Immunocytochemistry
For all the antibodies the procedure is as follows. 5 &mgr;m sections are first treated by antigen retrieval techniques, together with microwaving, in citrate buffer (pH 6.0).
Exposure is for 20 minutes and the slides left to cool down in the buffer. Where the antibodies are goat polyclonal, the peroxidase anti-peroxidase (PAP) method with diaminobenzidine (DAB) as chromogen is utilised, otherwise for rabbit polyclonals the APAAP system with new Fuchsin is used. The sections are first blocked with 1% bovine serum albumen (BSA) for 30 minutes to block non-specific receptors, followed by incubation with the primary antibody for 30 minutes at room temperature. Each antibody is titrated out and the optimal dilutions are as follows:
Antibodies Used
Lung Tissue
Adrenal
AT
1
Clone N 10
1:200
1:500
Clone N 306
1:200
1:500
AT
2
Clone C 18
1:150
1:500
As a negative control, for the rabbit polyclonal a negative serum from Dako (Prosan, Ghent, Belgium) is used, for the goat polyclonal the primary antibody is omitted.
ISH
5 &mgr;m sections are deparaffinised and then exposed to ‘in situ’ hybridisation follow

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