WO2003037373A1 - Utiilisation d'un antagoniste du recepteur ep2 ou ep4 et/ou d'un inhibiteur de la cox-1 pour traiter un cancer cervical - Google Patents

Utiilisation d'un antagoniste du recepteur ep2 ou ep4 et/ou d'un inhibiteur de la cox-1 pour traiter un cancer cervical Download PDF

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WO2003037373A1
WO2003037373A1 PCT/GB2001/004826 GB0104826W WO03037373A1 WO 2003037373 A1 WO2003037373 A1 WO 2003037373A1 GB 0104826 W GB0104826 W GB 0104826W WO 03037373 A1 WO03037373 A1 WO 03037373A1
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cox
expression
cervix
cells
inhibitor
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PCT/GB2001/004826
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English (en)
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Henry Nicolas Jabbour
Kurt Jason Sales
Arieh Katz
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Medical Research Council
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
    • A61K31/19—Carboxylic acids, e.g. valproic acid
    • A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
    • A61K31/196—Carboxylic acids, e.g. valproic acid having an amino group the amino group being directly attached to a ring, e.g. anthranilic acid, mefenamic acid, diclofenac, chlorambucil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/403—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with carbocyclic rings, e.g. carbazole
    • A61K31/404—Indoles, e.g. pindolol
    • A61K31/405—Indole-alkanecarboxylic acids; Derivatives thereof, e.g. tryptophan, indomethacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
    • A61K31/407—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil condensed with other heterocyclic ring systems, e.g. ketorolac, physostigmine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A61K31/4152—1,2-Diazoles having oxo groups directly attached to the heterocyclic ring, e.g. antipyrine, phenylbutazone, sulfinpyrazone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4196—1,2,4-Triazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/54—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame
    • A61K31/5415—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one sulfur as the ring hetero atoms, e.g. sulthiame ortho- or peri-condensed with carbocyclic ring systems, e.g. phenothiazine, chlorpromazine, piroxicam
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/557—Eicosanoids, e.g. leukotrienes or prostaglandins
    • A61K31/5575—Eicosanoids, e.g. leukotrienes or prostaglandins having a cyclopentane, e.g. prostaglandin E2, prostaglandin F2-alpha
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present invention relates to a method of treating a disease, and in particular to a method of treating a neoplastic condition of the cervix.
  • Cervical cancer is one of the leading causes of cancer-related death in women world-wide. Cervical cancer is particularly common in less developed countries, including South and Central America, Southeast Asia and Sub-Saharan Africa. Histopathologic classification of cervical cancer precursors is generally using the GIN nomenclature (cervical intraepithelial neoplasia).
  • the nomenclature GIN grades I to III are used to describe pre- invasive epithelial lesions or various categories of dysplasia and carcinoma in situ.
  • CIN grade I is equivalent to mild dysplasia in which undifferentiated cells occupy approximately the lower one third of the epithelium.
  • CIN grade II is equivalent to moderate dysplasia where undifferentiated cells replace two thirds of the thickness of normal epithelium.
  • CIN grade III denotes severe dysplasia and carcinoma in situ.
  • Symptoms of cervical carcinoma include vaginal bleeding, persistent sero- sanguinous foul-smelling discharge, disruption of normal bladder or bowel function and leg oedema.
  • the WHO recognises three general categories of cervical cancer, namely squamous cell carcinoma, adenocarcinoma and other epithelial tumours including less common types such as adenosquamous carcinoma, glassy cell carcinoma, adenoid basal cell carcinoma as well as carcinoid-like and small cell carcinoma.
  • Adenocarcinomas and to a lesser extent adenosquamous carcinomas and rarely sarcomas, lymphoma and melanoma account for approximately 20% of the invasive cervical carcinoma and display a variety of types and subtypes.
  • Cyclooxygenases (sometimes called prostaglandin endoperoxide synthase) are involved in prostaglandin synthesis. Two cyclooxygenase isoenzymes, COX-1 and COX-2, have been identified. COX-1 expression is considered to be constitutive, as basal levels of COX-1 mRNA and protein are observed to be present and generate prostaglandins for normal physiological functions. In contrast, COX-2 expression is inducible.
  • COX-2 expression is high in stage IB cervical cancer whereas COX-1 was expressed without regard to location of the tumour cells or type of cancer cell and the authors indicate that COX-1 is unrelated to apoptosis, tumourigenesis and tumour invasion mechanisms.
  • Kulkarni et al (2001) Clin. Cancer Res. 7, 429-434 indicates that COX-2 is overexpressed in human cervical cancer.
  • Prostaglandin E2 elicits its autocrine/paracrine effects on target cells through interaction with transmembrane G protein coupled receptors.
  • PGE 2 receptors four main sub-types of PGE 2 receptors have been identified based on responses to agonists and antagonists and are pharmacologically divided into EP1, EP2, EP3 and EP4 which utilise alternate and in some cases opposing intracellular signalling pathways.
  • EP2 and EP4 increase cAMP levels via G ⁇ s .
  • the inventors have surprisingly found that COX-1 is upregulated in cervical carcinoma as are the EP2 and EP4 receptors, and basal cAMP levels are significantly higher in cervical carcinoma tissues compared to normal cervix.
  • the inventors propose new methods of treating a neoplastic condition of the cervix, by which we include CIN grades I to III, carcinoma in situ and invasive carcinoma.
  • a first aspect of the invention provides a method of treating a neoplastic condition of the cervix in a patient the method comprising administering to the patient an inhibitor of cyclooxygenase- 1 (COX-1).
  • COX-1 cyclooxygenase- 1
  • a second aspect of the invention provides a method of treating a neoplastic condition of the cervix in a patient the method comprising administering to the patient an EP2 or EP4 receptor antagonist.
  • a neoplastic condition of the cervix we specifically include CIN grades I to III, carcinoma in situ and invasive carcinoma.
  • the carcinoma may be at any stage of differentiation.
  • the cervix is the uterine cervix.
  • the method of the invention is used to treat adenocarcinoma or squamous cell carcinoma of the cervix.
  • the patient may be any patient who is suffering from a neoplastic condition of the cervix or a patient who is at risk from this. Risk factors include human papilloma virus infection (although 20% of women developing cervical carcinoma do not have HPV infection), smoking, having more than one sexual partner and a family history of the disease.
  • the patient to be treated may be any female individual who would benefit from such treatment. Typically and preferably the patient to be treated is a human female.
  • the methods of the invention may be used to treat female mammals, such as the females of the following species: cows, horses, pigs, sheep, cats and dogs. Thus, the methods have uses in both human and veterinary medicine.
  • women at risk are treated, for example, women with HPV infection and/or who have a family history of cervical cancer.
  • the inhibitor of COX-1 may be any suitable inhibitor of COX-1.
  • suitable we mean that the inhibitor may be administered to the patient. It will be appreciated that one or more inhibitors of COX-1 may be administered to the patient.
  • Non-steroidal anti-inflammatory drugs typically are inhibitors of COX-1. Many NSAIDs are known in the art, and include those disclosed in Martindale's, The complete drug reference, 32 nd Edition, Parfitt, K (ed), Pharmaceutical Press, London, UK, incorporated herein by reference.
  • NSAIDs include ibuprofen, naproxen, fenbufen, fenoprofen, flurbiprofen, ketoprofen, dexketoprofen, tiaprofenic acid, azapropazone, diclofenac, aceclofenac, diflunisal, etodolac, indomethacin, ketorolac, mefenamic acid, meloxicam, namubetone, phenylbutazone, piroxicam, sulindac, tenoxicam and tolfenamic acid.
  • the inhibitor is selective for COX-1 over COX-2.
  • selective we mean that the inhibitor inhibits COX-1 at least ten times more effectively than COX-2.
  • the prostaglandin EP2 receptor antagonist may be any suitable EP2 receptor antagonist.
  • the prostaglandin EP4 receptor antagonist may be any suitable EP4 receptor antagonist.
  • suitable we mean that the antagonist is one which may be administered to the patient.
  • the receptor antagonists are molecules which bind to their respective receptors, compete with the natural ligand (PGE 2 ) and inhibit the initiation of the specific receptor-mediated signal transduction pathways.
  • the receptor antagonists are typically selective to the particular receptor and typically have a higher binding affinity to the receptor than the natural ligand. Although antagonists with a higher affinity for the receptor than the natural ligand are preferred, antagonists with a lower affinity may also be used, but it may be necessary to use these at higher concentrations.
  • the antagonists bind reversibly to their cognate receptor.
  • antagonists are selective for a particular receptor and do not affect the other receptor; thus, typically, an EP2 receptor antagonist binds the EP2 receptor but does not substantially bind the EP4 receptor, whereas an EP4 receptor antagonist binds the EP4 receptor but does not substantially bind the EP2 receptor.
  • the EP2 or EP4 receptor antagonist is selective for the particular receptor subtype. By this is meant that the antagonist has a binding affinity for the particular receptor subtype which is at least ten-fold higher than for at least one of the other EP receptor subtypes.
  • selective EP4 receptor antagonists have at least a ten-fold higher affinity for the EP4 receptor than any of the EP1, EP2 or EP4 receptor subtypes.
  • EP2 receptor antagonists include AH6809 (Pelletier et al (2001) Br. J. Pharmacol. 132, 999-1008).
  • EP4 receptor antagonists include AH23848B (developed by Glaxo) and AH22921X (Pelletier et al (2001) Br. J. Pharmacol. 132, 999-1008.
  • the chemical name for AH23848B is ([lalpha(z), 2beta5al ⁇ ha]-(+/-)-7-[5- [[(1,1 '-biphenyl)-4-yl]methoxy]-2-(4-morph olinyl)-3-oxo-cyclopentyl]-4- heptenoic acid) (see Hillock & Grankshaw (1999) Eur. J. Pharmacol. 28, 99-108).
  • EP4RA Li i (2000) Endocrinology 141, 2054-61 is an EP(4) - selective ligand (Machwate et al (2001) Mol. Pharmacol. 60:36-41).
  • the omega-substituted prostaglandin E derivatives described in WO 00/15608 (EP 1 114 816) (Ono Pharm Co Ltd) bind EP4 receptors selectively and may be EP4 receptor antagonists.
  • EP4 receptor antagonists are also described as EP4 receptor antagonists, as are some of the compounds described in WO 00/18744 (Fujisawa Pharm Co Ltd).
  • the 5-thia-prostaglandin E derivatives described in WO 00/03980 (EP 1 097 922) (Ono Pharm Co Ltd) may be EP4 receptor antagonists.
  • EP4 receptor antagonists are also described in WO 01/10426 (Glaxo), WO 00/21532 (Merck) and GB 2 330 307 (Glaxo).
  • EP4 receptor antagonists WO 00/21532 describes the following as EP4 receptor antagonists:
  • GB 2 330 307 describes [l ⁇ (Z), 2 ⁇ ,5 ]-(+)-7-[5-[[(l,l '-biphenyl)-4- yl]methoxy]-2-(4-morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid and [lR[l (z),2 ⁇ ,5 ⁇ ]]-(-)-7-[5-[[(l,l '-biphenyl)-4-yl]methoxy]-2-(4- morpholinyl)-3 -oxocy clopentyl] -4-heptenoic acid.
  • WO 00/18405 (Phar agene) describes the EP4 receptor antagonists AH22921 and AH23848 (which are also described in GB 2 028 805 and US 4, 342, 756).
  • WO 01/72302 (Pharmagene) describes further EP4 receptor antagonists, for example those described by reference to, and included in the general formula (I) shown on page 8 et seq.
  • EP2 receptor antagonists may be administered to the patient. It will also be appreciated that a combination of one or more EP2 or EP4 receptor antagonists may be administered to the patient.
  • the patient is administered one or more inhibitors of COX-1 and one or more EP2 or EP4 receptor antagonists. It is particularly preferred that a combination of one or more inhibitors of COX- 1 and one or more EP2 or EP4 receptor antagonists is used in the treatment. Treatment with an EP2 and/or EP4 receptor antagonist in the absence of a COX-1 inhibitor may leave further effects of elevated expression of COX-1 untreated, whereas treatment with a COX-1 inhibitor alone may leave the undesirable effects of elevated EP2 and/or EP4 receptor untreated.
  • the invention includes the administration to the patient of a combination of one or more inhibitors of COX-1 and one or more EP2 or EP4 receptor antagonists.
  • the inhibitors of COX-1, or the EP2 or EP4 receptor antagonists, or combinations thereof, are typically administered in an effective amount to combat the neoplastic condition of the cervix.
  • the treatment agent or agents may be used to alleviate symptoms (ie are used palliatively) or may be used to treat the condition.
  • treatment agent or therapeutic agent we mean the inhibitor of COX-1 or EP” or EP4 receptor antagonist as the context demands).
  • the method may also be used prophylactically, for example to prevent the development of a neoplastic lesion or to prevent transition from one early stage to. a later stage, or by reversing early changes either partially or completely, such as at CIN stages I, II or III.
  • treating specifically includes prophylactic treatment.
  • the treatment agent or agents may be administered by any suitable route, and in any suitable form. It is desirable to administer an amount of the inhibitor of COX-1, or EP2 or EP4 receptor antagonist, or combination thereof, that is effective in alleviating or ameliorating or curing or preventing the neoplastic condition of the cervix.
  • the treatment agents When more than one of the treatment agents are administered to the patient, it will be appreciated that they can be administered contemporaneously or sequentially. In addition, it may be desirable to administer the agents in different forms or by different routes of administration or to different parts of the patient's body.
  • a third aspect of the invention provides the use of an inhibitor of COX-1 in the manufacture of a medicament for treating a neoplastic condition of the cervix in a patient.
  • the inhibitor of COX-1 may be administered alone, it may desirable if the patient is also administered one or more antagonists of an EP2 or EP4 receptor.
  • the patient is also administered we include that the patient has been, is being or will be administered the said further therapeutic agent or agents.
  • a fourth aspect of the invention provides the use of an EP2 or EP4 receptor antagonist in the manufacture of a medicament for treating a neoplastic condition of the cervix in a patient.
  • the EP2 or EP4 receptor antagonist may be administered alone, it may desirable if the patient is also administered one or more inhibitors of COX-1.
  • the patient is also administered we include that the patient has been, is being or will be administered the said further therapeutic agent or agents.
  • a fifth aspect of the invention provides the use of any one or more of (a) an inhibitor of COX-1 and (b) an EP2 or EP4 receptor antagonist for treating a neoplastic condition of the cervix.
  • a compound of the invention While it is possible for a compound of the invention to be administered alone or in a "naked” form, it is preferable to present it as a pharmaceutical formulation, together with one or more acceptable carriers.
  • the carrier(s) must be “acceptable” in the sense of being compatible with the compound of the invention and not deleterious to the recipients thereof.
  • the carriers will be water or saline which will be sterile and pyrogen free.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active ingredient (compound of the invention) with the carrier which constitutes one or more accessory ingredients. In general the formulations are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product.
  • Formulations in accordance with the present invention suitable for oral administration may be presented as discrete units such as capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; as a powder or granules; as a solution or a suspension in an aqueous liquid or a non-aqueous liquid; or as an oil-in-water liquid emulsion or a water-in- oil liquid emulsion.
  • the active ingredient may also be presented as a bolus, electuary or paste.
  • a tablet may be made by compression or moulding, optionally with one or more accessory ingredients.
  • Compressed tablets may be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as a powder or granules, optionally mixed with a binder (eg povidone, gelatin, hydroxypropylmethyl cellulose), lubricant, inert diluent, preservative, disintegrant (eg sodium starch glycolate, cross-linked povidone, cross-linked sodium carboxymethyl cellulose), surface-active or dispersing agent.
  • Moulded tablets may be made by moulding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent.
  • the tablets may optionally be coated or scored and may be formulated so as to provide slow or controlled release of the active ingredient therein using, for example, hydroxypropylmethylcellulose in varying proportions to provide desired release profile.
  • COX-1 inhibitors are known to have adverse effects in the stomach where, for example, under certain conditions they may be involved in forming ulcers. It is preferred that if the COX-1 inhibitor is administered orally that it is present in formulation which is designed to limit such possible adverse effects.
  • formulations are well known in the art and include, for example, enteric coated tablets wherein the coating is resistant to the acid conditions in the stomach (and so the COX-1 inhibitor is not released in the stomach) but is not resistant to the alkali conditions in the small intestines where the COX-1 inhibitor may be released.
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.
  • buccal administration is also preferred.
  • Formulations suitable for parenteral administration include aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose or an appropriate fraction thereof, of an active ingredient.
  • formulations of this invention may include other agents conventional in the art having regard to the type of formulation in question, for example those suitable for oral administration may include flavouring agents.
  • EP2 and EP4 receptor antagonists are proteins or peptides. Proteins and peptides may be delivered using an injectable sustained-release drug delivery system. These are designed specifically to reduce the frequency of injections.
  • An example of such a system is Nutropin Depot which encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that, once injected, release rhGH slowly over a sustained period.
  • the protein and peptide can be administered by a surgically implanted device that releases the drug directly to the required site.
  • Vitrasert releases ganciclovir directly into the eye to treat CMV retinitis.
  • the direct application of this toxic agent to the site of disease achieves effective therapy without the drug's significant systemic side-effects.
  • Electroporation therapy (EPT) systems can also be employed for the administration of proteins and peptides.
  • EPT Electroporation therapy
  • a device which delivers a pulsed electric field to cells increases the permeability of the cell membranes to the drug, resulting in a significant enhancement of intracellular drug delivery.
  • Proteins and peptides can be delivered by electroincorporation (El).
  • El occurs when small particles of up to 30 microns in diameter on the surface of the skin experience electrical pulses identical or similar to those used in electroporation. In El, these particles are driven tlirough the stratum corneum and into deeper layers of the skin.
  • the particles can be loaded or coated with drugs or genes or can simply act as "bullets" that generate pores in the skin through which the drugs can enter.
  • ReGel injectable system An alternative method of protein and peptide delivery is the ReGel injectable system that is thermo-sensitive. Below body temperature, ReGel is an injectable liquid while at body temperature it immediately forms a gel reservoir that slowly erodes and dissolves into known, safe, biodegradable polymers. The EP2 or EP4 receptor antagonist is delivered over time as the biopolymers dissolve. Protein and peptide pharmaceuticals can also be delivered orally.
  • the process employs a natural process for oral uptake of vitamin B 12 in the body to co-deliver proteins and peptides. By riding the vitamin B 12 uptake system, the protein or peptide can move through the intestinal wall. Complexes are synthesised between vitamin B 12 analogues and the drug that retain both significant affinity for intrinsic factor (IF) in the vitamin B ]2 portion of the complex and significant bioactivi y of the drug portion of the complex.
  • IF intrinsic factor
  • Proteins and polypeptides can be introduced to cells by "Trojan peptides". These are a class of polypeptides called penetratins which have translocating properties and are capable of carrying hydrophilic compounds across the plasma membrane. This system allows direct targeting of oligopeptides to the cytoplasm and nucleus, and may be non-cell type specific and highly efficient. See Derossi et al (1998), Trends Cell Biol 8, 84-87.
  • the treatment agent (or combination thereof) is administered at a dose (or in multiple doses) which produces a beneficial therapeutic effect in the patient. Suitable doses may be determined by the physician.
  • the dose to be administered is determined upon age, body weight, mode of administration, duration of the treatment, and pharmacokinetic and toxicological properties of the treatment agents.
  • the treatment agent is administered orally. It is further preferred if the treatment agent (or combination thereof) is administered to the female reproductive system.
  • the treatment agent may suitably be administered intravaginally using, for example, a gel or cream or vaginal ring or tampon.
  • the treatment agent (or combination thereof) may also advantageously be administered using an intrauterine device, although this route of administration is less preferred for carcinoma in situ and invasive carcinoma (but may be used for treating at CIN grades I, II or III).
  • the gel or cream is one which is formulated for administration to the vagina. It may be oil based or water based.
  • the treatment agent or combination thereof is present in the cream or gel in a sufficient concentration so that an effective amount is administered in a single (or in repeated) application.
  • the vaginal ring comprises a polymer which formed into a "doughnut" shape which fits within the vagina.
  • the treatment agent (or combination thereof) is present within the polymer, typically as a core, which may dissipate through the polymer and into the vagina and/or cervix in a controlled fashion.
  • Vaginal rings are known in the art.
  • the vaginal ring may be disposable; alternatively, the vaginal ring may be used over a time interval of around three months to one year, during which time sufficient treatment agent (or combination thereof) is released to have a beneficial effect over that period of time.
  • the polymer from which the ring is made, the size and shaper of the ring and the content of antagonist, as well as other parameters may be selected by reference to how long the ring is in situ in the vagina.
  • the tampon is impregnated with the treatment agent (or combination thereof) and that a sufficient amount of the freatment agent (or combination thereof) is present in the tampon bearing in mind that the tampon is generally used for a single day.
  • the intrauterine device is for placing in the uterus over extended periods of time, such as between one and five years.
  • the intrauterine device comprises a plastic frame, often in the shape of a "T”and contains sufficient freatment agent (or combination thereof) to be released over the period of use.
  • the freatment agent (or combination thereof) is generally present within or encompassed by a slow-release polymer which forms part of the device, such as in the form of a "sausage" of antagonist which wraps around the long arm of the "T" which is typically covered with a confrolled-release membrane.
  • Intrauterine devices are known in the art.
  • a sixth aspect of the invention provides a therapeutic system for treating a neoplastic condition of the cervix the system comprising a combination of any two or more of (a) an inhibitor of COX-1 and (b) an EP2 or EP4 receptor antagonist.
  • the particular freatment agents in the therapeutic system may be selected according to the preferences given above.
  • the therapeutic system may be purchased ready to use, or it may be assembled by the pharmacist or physician.
  • a seventh aspect of the invention provides a therapeutic system for treating a neoplastic condition of the cervix the system comprising any one or more of (a) an inhibitor of COX-1 and (b) an EP2 or EP4 receptor antagonist adapted for delivery to the cervix. Suitable adaptations for delivery of the freatment agent to the cervix are described above.
  • FIG. 1 Relative expression of COX-1 and COX-2 RNA in cervical squamous cell carcinoma (C1-C14), adenocarcinoma (C15-C18) and normal cervix (N1-N8) as determined by real-time quantitative RT-PCR.
  • B Western blot analysis of 50 ⁇ g total protein isolated from human cervical carcinoma tissue. The proteins were loaded onto a 10% SDS-gel, elecfrophoresed and subsequently transferred to PVDF membrane. The immunoblot was probed with antibody raised against the C-terminus of human COX-1 or COX-2.
  • FIG. 1 Localisation of COX-1 expression in epithelial cells of squamous cell carcinomas and columnar and glandular epithelium of adenocarcinomas (A and C respectively). Minimal COX-1 signal was detected in normal cervical tissue (E). Sections that were stained with pre- adsorbed COX-1 sera are shown in B, D and F for squamous cell carcinoma, adenocarcinoma and normal cervix respectively (negative controls). Scale bar is 100 ⁇ m.
  • FIG. 3 (A) Western blot analysis of 20 ⁇ g total protein isolated from wild-type HeLa Tet-Off and HeLa COX-1 Tet-Off cells grown for 24, 48 and 72 hrs respectively in the absence of DOX. In parallel confrol uninduced HeLa COX-1 Tet-Off and wild-type HeLa Tet-Off cells were maintained for 72 hrs under the same conditions supplemented daily with DOX to a final concentration of 1 ⁇ g/ml. The proteins were loaded onto a 4-20% SDS-gel, elecfrophoresed and subsequently transferred to PVDF membrane. The immunoblot was probed with antibody raised against the C-terminus of human COX-1. A specific band of approximately 72kDa was detected.
  • COX-1 was normalised for protein loading against ⁇ -actin on the same blot.
  • B The functionality of the transfected COX-1 cDNA was assessed by ELISA, by measuring PGE2 secretion into the culture medium following COX-1 induction in the presence or absence of the COX-enzyme inhibitor indomethacin, and treatment of HeLa cells with 5 ⁇ g/ml arachidonic acid.
  • FIG. 4 Western blot analysis of 20 ⁇ g of total clarified cell lysate isolated from HeLa COX-1 Tet-Off cells grown for 72 hrs in the presence of DOX (uninduced) or 24, 48 and 72 hrs respectively in the absence of DOX to induce COX-1 expression.
  • A Expression of COX-2 and PGES was induced coincident with COX-1 overexpression in HeLa cells.
  • B Cotreatment of HeLa cells with indomethacin abolished the COX-1 -mediated up-regulation of COX-2 and PGES.
  • C Partial inhibition of the COX-1- mediated up-regulation of COX-2 and PGES expression was observed following co-treatment with the selective COX-2 inhibitor NS-398. Proteins were normalised for loading against ⁇ -actin on the same blot.
  • FIG. 1 Fold induction of expression of PGE2 receptors (EP1 - EP4) in HeLa COX-1 Tet-Off cells as determined by real-time quantitative RT- PCR. COX-1 expression was induced for 24, 48 and 72 hrs in the presence or absence of the COX enzyme inhibitor indomethacin. Fold induction was determined by dividing the relative expression in induced cells by the relative expression in uninduced cells.
  • FIG. 7 Western blot analysis of 20 ⁇ g of total clarified cell lysate isolated from HeLa COX-1 Tet-Off cells grown for 72 hrs in the presence of DOX or 24, 48 and 72 hrs in the absence of DOX to induce COX-1 expression.
  • A Immunoblot of bFGF expression following DOX withdrawal from the culture medium. bFGF expression was induced coincident with COX-1 overexpression. Up-regulated bFGF expression was abolished by indomethacin and partially inhibited by NS-398.
  • B Immunoblot of VEGF expression following DOX withdrawal from the culture medium. VEGF was induced after 72 hrs of COX-1 overexpression.
  • VEGF expression was abolished by indomethacin and partially inhibited by NS-398.
  • C Immunoblot of Ang-1 expression following DOX withdrawal from the culture medium. Ang-1 was induced coincident with COX-1 overexpression after 48 hrs. Upregulated Ang-1 expression was abolished by indomethacin and partially inhibited by NS-398.
  • D Immunoblot of Ang-2 expression following DOX withdrawal from the culture medium. Ang-1 was induced after 48 hrs of COX-1 overexpression. Up-regulated Ang-2 expression was abolished by indomethacin and partially inhibited by NS-398. Proteins were normalised for loading against ⁇ -actin.
  • FIG. 8 (A) Relative expression of COX-2 RNA in cervical squamous cell carcinoma (C1-C8), adenocarcinoma (C9 and CIO) and normal cervix (N1-N5) as determined by real-time quantitative RT-PCR.
  • B Western blot analysis of 100 ⁇ g total protein isolated from human cervical carcinoma tissue. The proteins were loaded onto a 10% SDS-gel, elecfrophoresed and subsequently transferred to PVDF membrane. The immunoblot was probed with antibody raised against the C-terminus of human COX-2. A specific band of approximately 72 kDa was detected in all squamous (C1-C8) and adenocarcinoma (C9 and CIO). No signal was detected in normal cervical tissue (N) (a representative sample is shown). Moreover, preadsorbing the antibody with the blocking peptide (B) abolished the COX-2 signal in all carcinoma samples (a representative sample is shown).
  • FIG. 9 COX-2 expression and PGE2 synthesis ( Figures a and b respectively) are detected in epithelial cells of squamous cell carcinoma and columnar and glandular epithelium of adenocarcinomas ( Figures c and d respectively). Minimal COX-2 and PGE2 signal was detected in normal cervical tissue ( Figures e and f respectively).
  • Figures g and h respectively are representative sections of adenocarcinoma incubated with COX-2 antibody preadsorbed with the blocking peptide (COX-2 negative confrol) and non-immune rabbit serum (PGE2 negative confrol). Scale bar is 100 ⁇ m.
  • FIG. 10 COX-2 ( Figure a) expression and PGE2 (Figure b) synthesis are detected in endothelial cells (arrowed) of all carcinoma tissues.
  • Vascular endothelial cells in cervical cancer tissues were localised using antibodies raised against the human CD34 endothelial cell marker ( Figure c).
  • Figure d is a representative section incubated with non-immune goat serum (CD34 negative confrol). Scale bar is 50 ⁇ m.
  • FIG. 11 Relative expression of EP2 (empty bars) and EP4 (solid bars) receptors in cervical squamous cell carcinoma (C1-C7), adenocarcinoma (C8) and normal cervix (N1-N5) as determined by real-time quantitative RT-PCR.
  • B cAMP response (pmol cAMP/mg protein) in squamous cell carcinoma (C1-C6), adenocarcinoma (C7) and normal cervix (N1-N5). Cervical tissues were treated with indomethacin overnight and either stimulated with 300 nM PGE2 (empty bars) or 50 ⁇ M forskolin (solid bars; positive confrol) or left unstimulated (grey bars).
  • Cyelooxygenase-1 is up-regulated in cervical carcinomas: autocrine/paracrine regulation of cyclooxygenase-2, PGE receptors and angiogenic factors by cyclooxygenase-1
  • COX-1 cyclooxygenase-1
  • RNA and protein in squamous cell carcinomas and adenocarcinoma of the cervix.
  • COX-1 expression in all carcinoma tissues was associated with enhanced expression of COX-2 RNA and protein.
  • the site of COX-1 expression was localised by immunohistochemistry to the neoplastic epithelial cells in all squamous cell carcinomas and adenocarcinomas studied. Minimal COX-1 immunoreactivity was detected in normal cervix.
  • COX-1 up-regulation To explore events associated with COX-1 up-regulation, we developed a doxycycline (DOX)-regulated expression system in HeLa (cervical carcinoma) cells.
  • DOX doxycycline
  • Overexpression of COX-1 in HeLa cells resulted in induced expression of cyclooxygenase-2 (COX-2) and prostaglandin E synthase (PGES) concomitant with increased prostaglandin E2 (PGE2) synthesis.
  • COX-2 cyclooxygenase-2
  • PGES prostaglandin E synthase
  • Treatment of HeLa cells overexpressing COX-1 with the dual COX-enzyme inhibitor indomethacin or selective COX-2 inhibitor NS-398 significantly reduced PGE2 synthesis.
  • COX-1 angiogenic factor
  • basic fibroblast growth factor bFGF
  • VEGF vascular endothelial growth factor
  • Ang-1 angiopoietin-1
  • Ang-2 angiopoietin-2
  • Uterine cervical cancer is considered an important clinical problem in developing countries, with high incidence of invasive disease reported for South African women (1).
  • Three histological categories of epithelial tumours of the cervix are recognised by the World Health Organisation (2). These are squamous cell carcinoma, adenocarcinoma and other less common types of epithelial tumours.
  • the most common histological type of cervical carcinoma is squamous cell carcinoma that accounts for 60%- 80% of all cervical cancers.
  • Adenocarcinoma accounts for less than 20% of invasive cervical carcinomas.
  • Numerous studies have demonstrated that epithelial tumours may be regulated by cyclooxygenase (COX)-enzyme products (3-7).
  • COX cyclooxygenase
  • COX-1 and COX-2 Two distinct isoforms of the COX enzyme, COX-1 and COX-2 have been reported (8-10). The relative contributions of COX-1 and/or COX-2-derived products in mediating events associated with cervical neoplasia remain to be elucidated. COX-1 expression is considered to be constitutive and generate prostaglandins for normal physiological functions (4, 11, 12). Transcription of COX-2 RNA and protein are upregulated in several epithelial carcinomas (3, 12-15) including carcinomas of the cervix (16-18). This has prompted the suggestion that the increased level of prostaglandins and other eicosanoids present in cancer tissue is a consequence of induced COX-2. More recently however, it has been demonstrated that both COX isoforms are inducible.
  • COX-1 levels are induced during differentiation (19, 20). COX-1 expression can be induced in vitro by vascular endothelial growth factor (VEGF) (21), arachidonic acid, forskolin, dibutryl-cAMP and prostaglandin E2 (PGE2) (22). In addition, elevated COX-1 expression has been reported in mouse lung tumours (23), human breast cancer (24) and human prostate carcinoma (25). These data suggest that both COX enzymes and/or their products may function in promoting and maintaining the neoplastic state.
  • VEGF vascular endothelial growth factor
  • PGE2 prostaglandin E2
  • COX catalyse the double oxygenation and reduction of arachidonic acid, following its release from membrane glycerophospholipids by phospholipase A2, to the intermediate form prostaglandin H2.
  • This intermediate serves as the substrate for terminal prostanoid synthases, which produce their specific prostaglandins such as PGE2 being synthesised by prostaglandin E synthase (PGES) (26- 28).
  • PGE2 has been shown to stimulate gene transcription (29), influence mitogenesis of normal human bone cells (30) and promote growth and metastasis of tumours (31).
  • the PGES antibody raised against the microsomal glutathione-dependent inducible PGES (27) was purchased from Caymen Chemical Co. (Caymen Chemical Co., Cheshire, UK); anti- goat- alkaline phosphatase (AP), anti-rabbit- AP, cloning cylinders, G418, hygromycin (Hyg), doxycycline (DOX) and indomethacin were purchased from Sigma (Sigma Chemical Company, Dorset, UK). Samples and synthetic standards for the PGE2 ELISA were purchased from Applied
  • Therapeutics (Applied Therapeutics, Paisley, UK) and NS-398 was purchased from Calbiochem (Calbiochem, Nottingham, UK). HeLa Tet-Off cells and Tet-system-approved foetal bovine serum were purchased from Clontech (Clontech, Hampshire, UK). Dulbecco's modified Eagle's medium nutrient mixture F-12 was purchased from Life Technologies (Gibco, Life Technologies, Paisley, UK) and penicillin-streptomycin was purchased from PAA (PAA Laboratories Ltd., Middlesex, UK). ECF chemiluminescence system was purchased from Amersham (Amersham, Little Chalfont, Bucks, UK).
  • Histologically normal cervical samples were obtained from patients undergoing Wertheims hysterectomy for non-malignant conditions.
  • Pathological typing was defined according to the International Federation of Obstetricians and Gynaecologists (37) staging upon physical examination. The ages of the patients ranged from 29 years to 80 years with a median age of 50 years. The study was approved by the University of Cape Town Research Ethics Committee and informed consent was obtained from all patients before tissue collection.
  • HeLa Tet-Off cells containing the regulatory plasmid were routinely maintained in Dulbecco's modified Eagle's medium nutrient mixture F-12 with glutamax-1 and pyridoxine, supplemented with 10% foetal bovine serum, lOO ⁇ g/ml G418 and 1% antibiotics (stock 500IU/ml penicillin and 500 ⁇ g/ml streptomycin) at 37°C and 5% C0 2 (v/v).
  • the Tet-Off expression system we used was developed by Gossen and co- workers (38) to deliver doxycycline-regulated expression based on the high specificity of the Escherichia coli tet repressor-operator-doxycycline interaction.
  • each clonal cell line is used as its own control (cells cultured in the presence of DOX) and the overexpression of the integrated target gene is modulated solely by removing DOX from the culture medium. This eliminates the need for a control clonal cell line transfected with vector alone (as used with constitutive stable expression systems) thereby overcoming the inherent variation that arises from different sites of integration of DNA between different clones.
  • HeLa Tet-Off cells containing the pTet-Off vector stably transfected and constitutively expressing the tetracycline-confrolled fransactivator tTA (composed of a fusion of the TetR and VP16 activation domain) were purchased from Clontech.
  • the pBS(SK-)/PSHI cDNA containing the full length COX-1 gene was used as the template plasmid.
  • the response plasmid pTRE2 (containing the minimal cytomegalovirus promoter containing Tet-operator sequences cloned upsfream of the cDNA to be expressed) and the plasmid for antibiotic selection (pTK-hygromycin) for use with the Tet-Off system were purchased from Clontech.
  • the COX-1 gene was excised from the template plasmid and ligated at the Bam HI site of the pTRE2 vector. The orientation of the insert was verified by dideoxy DNA sequencing using the sequence specific primers: 5'-CGCCTGGAGACGCCATCC-3' and 5'- CCACACCTCCCCCTGAAC-3' (Clontech).
  • telomeres were plated in 12 well dishes in complete medium containing lOO ⁇ g/ml G418 per well and were allowed to attach and grow overnight.
  • the pTRE2 vector containing the COX-1 gene (2 ⁇ g) was co-transfected with pTK-hygromycin (O.l ⁇ g, which contains the hygromycin gene under control of the minimal thymidine kinase (TK) promoter) into the HeLa Tet-Off cell line at about 80% confluency using pfx-5 (Invitrogen, De Schelp, Netherlands) diluted in Optimem (Gibco). Cells were incubated for 4 hrs at 37°C in 5% humidified C02- Thereafter the medium was replaced with fresh complete medium containing no G418.
  • TK-hygromycin O.l ⁇ g, which contains the hygromycin gene under control of the minimal thymidine kinase (TK) promoter
  • Clones were allowed to grow for 72 hrs. Transfected cells were then seeded together with wild-type cells. Clones were selected against 200 ⁇ g/ml hygromycin in the presence of 1 ⁇ g/ml DOX. At least 50 hygromycin-resistant clones were picked using cloning cylinders. Clones were allowed to grow under continuous selection with hygromycin in the presence of DOX and then screened for the ability to express COX-1 in the presence and absence of DOX by immunoblot analysis. Three clones with the greatest inducible overexpression of COX-1 (clone 1.2, 2.2 and 3.1) were selected for further experiments. All clones were characterised and exhibited identical phenotypic and biochemical alterations.
  • RNA samples were extracted from cervical tissue (squamous cell carcinomas, C1-C14; adenocarcinomas, C15-C18 and normal cervix, N1-N8) using Tri-Reagent (Sigma) as per the manufacturer's instruction.
  • cells (2 x 10 ) were seeded in 6 well plates, allowed to attach and grow overnight in the presence of DOX.
  • the cells were synchronised by incubating with serum-free medium for 24 hrs. Thereafter, the medium was replaced with fresh complete medium and COX-1 overexpression was induced by growing cells in medium containing no DOX. Control cells were maintained in DOX. Cells were harvested after 24, 48 and 72 hrs with 1 ml/well Tri-Reagent (Sigma) as per the manufacturer's protocol.
  • RNA samples were reverse transcribed using MgCl 2 (5.5mM), dNTPs (0.5mM each), random hexamers (1.25 ⁇ M), oligo-dT (1.25 ⁇ M), RNAase inhibitor (0.4U/ ⁇ l) and multiscribe reverse transcriptase (1.25U/ ⁇ l; all from PE Biosystems, Warrington, UK).
  • the mix was aliquoted into individual tubes (16 ⁇ l/tube) and template RNA was added (4 ⁇ l/tube of 250ng/ ⁇ l RNA). Samples were incubated for 60 mins at 25°C, 45 mins at 48°C and then at 95°C for 5 mins.
  • a reaction mix was made containing Taqman buffer (5.5mM MgCl 2 , 200 ⁇ M dATP, 200 ⁇ M dCTP, 200 ⁇ M dGTP, 400 ⁇ M dUTP), ribosomal 18S forward and reverse primers and probe (all at 50nM), forward and reverse primers for COX-1, COX-2, EP1, EP2, EP3 or EP4 receptor (300 nM), COX-1, COX-2, EP1, EP2, EP3 or EP4 receptor probe (200nM), AmpErase UNG (O.OlU/ ⁇ l) and AmpHTaq Gold DNA Polymerase (0.025U/ ⁇ l; all from PE Biosystems).
  • Taqman buffer 5.5mM MgCl 2 , 200 ⁇ M dATP, 200 ⁇ M dCTP, 200 ⁇ M dGTP, 400 ⁇ M dUTP
  • ribosomal 18S forward and reverse primers and probe all at 50nM
  • a volume of 48 ⁇ l of reaction mix was aliquoted into separate tubes for each cDNA sample and 2 ⁇ l/replicate of cDNA was added. After mixing, 23 ⁇ l of sample were added to the wells on a PCR plate. Each sample was added in duplicate. A no template confrol (containing water) was included in triplicate. Wells were sealed with optical caps and the PCR reaction run on an ABI Prism 7700 using standard conditions. COX-1, COX-2 and EP receptor primers and probe for quantitative PCR were designed using the PRIMER express program (PE Biosystems).
  • the sequence of the COX-1 primers and probe were as follows: Forward: 5'- TGT TCG GTG TCC AGT TGC AAT A-3'; Reverse: 5'- ACC TTG AAG GAG TCA GGC ATG AG -3'; Probe (FAM labelled): 5'- CGC AAC CGC ATT GCC ATG GAG T-3'.
  • the sequence of the COX-2 primers and probe were as follows: Forward: 5'-CCT TCC TCC TGT GCC TGA TG- 3'; Reverse: 5'-ACA ATC TCA TTT GAA TCA GGA AGG T-3'; Probe (FAM labelled): 5'- TGC CCG ACT CCC TTG GGT GTC A -3'.
  • the sequence of the EP1 receptor primers and probe were as follows: Forward: 5'- AGA TGG TGG GCC AGC TTG T-3'; Reverse: 5'- GCC ACC AAC ACC AGC ATT G -3'; Probe (FAM labelled): 5'- CAG CAG ATG CAC GAC ACC ACC ATG- 3'.
  • the sequence of the EP2 receptor primers and probe were as follows; Forward: 5'-GAC CGC TTA CCT GCA GCT GTA C-3'; Reverse: 5'-TGA AGT TGC AGG CGA GCA-3'; Probe (FAM labelled): 5'-CCA CCC TGC TGC TGC TTC TCA TTG TCT-3'.
  • the sequence of the EP3 receptor primers and probe were as follows; Forward: 5' -GAC GGC CAT TCA GCT TAT GG- 3'; Reverse: 5'- TTG AAG ATC ATT TTC AAC ATC ATT ATC A- 3'; Probe (FAM Labelled): 5' CTG TCG GTC TGC TGG TCT CCG CTC- 3'.
  • the sequence of the EP4 receptor primers and probe were as follows; Forward: 5'-ACG CCG CCT ACT CCT ACA TG-3'; Reverse: 5'-AGA GGA CGG TGG CGA GAA T-3'; Probe (FAM labelled): 5'-ACG GGG GCT TCA GCT CCT TCC T-3'.
  • the ribosomal 18S primers and probe sequences were as follows; Forward: 5' -CGG CTA CCA CAT CCA AGG AA-3'; Reverse: 5'-GCT GGA ATT ACC GCG GCT-3'; Probe (VIC labelled): 5'-TGC TGG CAC CAG ACT TGC CCT C-3 ⁇
  • Expression of COX-1 and EP receptors were normalised to RNA loading for each sample using the 18S ribosomal RNA as an internal standard.
  • Relative COX-1 and COX-2 expression in carcinoma tissue was calculated by dividing the expression in carcinoma tissue by the expression in normal cervix.
  • Relative expression of EP receptors was calculated, from 3 independent experiments, by dividing the expression in induced cells by expression in uninduced cells. The data are presented as mean + SEM.
  • COX-1 and COX-2 protein expression in cervical carcinomas and normal cervix was assessed by Western blotting.
  • Proteins were extracted from cervical tissue (squamous cell carcinomas, C19-C32; adenocarcinomas C33-C36 and normal cervix, N9-N16) by homogenisation in protein lysis buffer (1% Triton X-100, 150mM NaCl, lOmM Tris/HCl pH7.4, ImM EDTA, 0.1% SDS containing 2mM PMSF). Thereafter insoluble material was pelleted by centrifugation at 14000g for 20 mins at 4°C. The clarified lysate was removed to a new tube for protein quantification and SDS- PAGE.
  • the protein content in the supernatant fraction was determined using protein assay kits (Bio-Rad, Hemel Hempstead, UK). A total of 50 ⁇ g of protein was resuspended in 20 ⁇ l of sample buffer (125 mM Tris-HCl pH6.8, 4% SDS, 5% 2-mercaptoethanol, 20% glycerol and 0.05% bromophenol blue) boiled for 5 mins at 95°C and run on a 10% SDS- polyacrylamide gel prior to Western blotting.
  • sample buffer 125 mM Tris-HCl pH6.8, 4% SDS, 5% 2-mercaptoethanol, 20% glycerol and 0.05% bromophenol blue
  • Cells were seeded in 5cm dishes and allowed to attach overnight. The following day, the cells were synchronised by incubating with serum-free medium for 24 hrs. Thereafter, the medium was replaced with fresh complete medium and the cells were grown in the presence or absence of DOX for 24, 48 and 72 hrs respectively. In parallel, cells were co-treated with indomethacin or NS-398. Cells were harvested by lysing in protein lysis buffer (150mM NaCl, lOmM Tris-HCl pH 7.4, ImM EDTA, 1% Triton X-100, 0.1% SDS). The protein content in the supernatant fraction was determined as described above. The clarified cell lysates (20 ⁇ g) were denatured and elecfrophoresed on 4-20% Tris-Glycine gels (NOVEX, Invitrogen).
  • membranes were incubated overnight with either COX-1 (1 :500), COX-2 (1 :500), ⁇ -actin (1:500), PGES (1:250), bFGF (1:500), VEGF (1:500), Ang-1 (1:250) and Ang-2 (1 :250) specific antibodies.
  • membranes were subsequently incubated for 1 hr with rabbit anti-goat secondary antibody (for COX-1/2, ⁇ -actin, Ang-1/2, bFGF) at a dilution of 1:30000 or goat anti-rabbit secondary antibody (PGES, VEGF) at a dilution of 1:30000.
  • the site of COX-1 expression was localised in cervical tissues by immunohistochemistry using archival cervical blocks (squamous cell carcinomas, C37-C47; adenocarcinomas, C48-C58 and normal cervix, N19-N23) obtained from the Department of Anatomical Pathology, University of Cape Town, South Africa.
  • archival cervical blocks squamous cell carcinomas, C37-C47; adenocarcinomas, C48-C58 and normal cervix, N19-N23
  • TESPA adenocarcinomas, C48-C58 and normal cervix, N19-N23
  • Antigen retrieval was performed by pressure cooking for 2 mins in 0.0 IM sodium citrate pH6. Sections were blocked using either 5% normal rabbit serum diluted in TBS. Subsequently the tissue sections were incubated with polyclonal goat anti-COX-1 antibody (sc-1752; Autogenbioclear) at a dilution of 1:200 at 4°C for 18 hrs. Control tissue was incubated with goat anti-COX-1 antibody pre-adsorbed to blocking peptide (sc-1752p; Autogenbioclear) as per the manufacturer's protocol.
  • polyclonal goat anti-COX-1 antibody sc-1752; Autogenbioclear
  • tissue sections probed with the goat anti-human COX-1 primary antibody was incubated with biotinylated rabbit anti-goat secondary IgG antibody (Dako) at a dilution of 1:500 at 25°C for 40 mins. Thereafter the tissue sections were incubated with sfreptavidin-peroxidase complex (Dako) at 25°C for 20 mins. Colour reaction was developed by incubation with 3.3'-diaminobenzidine (Dako). The tissue sections were counterstained in aqueous hematoxylin, followed by sequential dehydration using graded ethanol and xylene, before mounting and coverslipping.
  • Dako biotinylated rabbit anti-goat secondary IgG antibody
  • HeLa COX-1 Tet-Off cells were seeded in 5cm dishes at a cell density of 5xl0 5 cells/dish and were allowed to grow and attach overnight. The following day, the cells were synchronised by incubating with serum-free medium for 24 hrs. COX-1 expression was induced for 24, 48 and 72 hrs respectively, by DOX withdrawal from the culture medium, in the presence or absence of indomethacin or NS-398. Arachidonic acid to a final concentration of 5 ⁇ g/ml was added to the culture medium following induction for 6 hrs. Thereafter, 1ml of medium was removed and added to lml of methyl oximating solution. Confrol uninduced cells were treated similarly but maintained with DOX supplemented daily.
  • PGE2 secretion into the culture medium was assayed by ELISA (39).
  • the ELISA was performed using 96 Well plates (Amine-binding plates; Costar, High Wycombe, UK) coated with donkey anti-rabbit antibody. Plates were then coated with rabbit immunoglobulin G (1 mg/ml diluted in PBS with 1% carbonate buffer, pH 9.6) at 200 ⁇ l/well for 16 hrs at 4°C. The solution was aspirated and blocking solution (50mM glycine, lOmg/ml bovine serum albumin) added at 25 ⁇ l/well for 2 hrs at 23°C.
  • blocking solution 50mM glycine, lOmg/ml bovine serum albumin
  • the plates were then washed and donkey anti-rabbit serum (Scottish Antibody Production Unit, Carluke, UK) added to a final volume of 150 ⁇ l/well, before washing, air drying and storage with desiccant at 4°C.
  • the link was prepared by ether extraction and reverse phase chromatography using 20mg of synthetic PGE2, 320 ⁇ l dry dimethylformamide, 3 ⁇ l butylchloroformate and 0.05mM biocytin.
  • Samples and synthetic standards were diluted in ELISA buffer (150mM NaCl, lOOmM Tris-HCl, 0.05% Tween-20, 50mM phenol red, ImM 2- methylisothiazolone, ImM bromonitrodioxane, 2mM EDTA, 2mg/ml bovine serum albumin to a final pH 7.2) and lOO ⁇ l of each added in duplicate to the plate.
  • the link was diluted l:1.5xl0 6 in ELISA buffer and 50 ⁇ l added to each well.
  • Antisera diluted 1:50000 in ELISA buffer, was added to a final volume of 50 ⁇ l to all wells except those used for measuring non-specific binding.
  • COX-1 and COX-2 in cervical carcinomas were investigated using real-time quantitative RT-PCR (Fig. IA) and Western blot analysis (Fig. IB). Expression of COX-1 and COX-2 RNA were significantly upregulated in 78% and 100% of cases of squamous cell carcinoma respectively and 100% of cases of adenocarcinoma investigated. By contrast, minimal COX-1 and COX-2 transcript were detected in normal cervical tissue by quantitative RT-PCR. COX-1 and COX-2 expression, as assessed by quantitative RT-PCR, was 19.9 ⁇ 5.9 and 118 + 32 fold greater in cervical carcinoma tissues than that observed in normal cervical tissue (P ⁇ 0.01).
  • COX-1 expression in the carcinoma tissue was investigated by immunohistochemistry. COX-1 expression was up-regulated in all carcinoma samples. COX-1 was localised to the neoplastically transformed squamous epithelium in squamous cell carcinoma (Fig. 2 A), and to neoplastically transfo ⁇ ned columnar epithelium lining the endocervical canal and the glandular epithelium of the endocervical glands in adenocarcinomas (Fig. 2C). Little or no immunoreactivity for COX-1 was observed in the normal cervical tissues (Fig. 2E). Pre-adsorbing the antibody with the blocking peptide (COX-1 negative confrol) abolished the COX-1 immunoreactivity in all carcinoma samples. Representative sections incubated with the blocking peptide are shown in Fig. 2B, 2D and 2F for squamous cell carcinoma, adenocarcinoma and normal cervical tissues respectively.
  • the functionality of the transfected COX-1 cDNA was assessed by measuring PGE2 secretion into the culture medium following COX-1 induction for 24, 48 and 72 hrs respectively.
  • a time-dependent increase in PGE2 secretion into the culture medium accompanied the induction of COX-1 expression.
  • PGE2 production was significantly elevated after 48 hrs (272.2 ⁇ 18.8nM; p ⁇ 0.05) and 72 hrs (537 ⁇ 22.5nM; p ⁇ 0.01) when compared with PGE2 levels in uninduced cells (118 ⁇ 6.75nM; Fig. 3B).
  • the addition of indomethacin reduced the PGE2 levels to 62 ⁇ 7nM and 76 ⁇ 0.7nM after 48 hrs and 72 hrs respectively (p ⁇ 0.01).
  • COX-enzyme products including PGE2 are known to induce COX-2 expression (22).
  • COX-1 Tet-Off HeLa cells were grown in the presence or absence of the dual COX-enzyme inhibitor indomethacin or the highly selective COX-2 inhibitor NS-398 for 24, 48 and 72 hrs. Following DOX withdrawal from the culture medium, a time-dependent increase in COX-1 overexpression was observed with maximal sustained overexpression after 72 hrs (Fig. 4A).
  • EP3 receptor franscript levels were significantly induced after 48 hrs of COX-1 overexpression (p ⁇ 0.05) compared with cells grown in the presence of indomethacin.
  • EP4 receptor franscript was significantly up-regulated after COX-1 overexpression for 24, 48 and 72 hrs compared with cells co- treated with the COX-enzyme inhibitor (p ⁇ 0.05).
  • COX-1 expression is up-regulated in human breast cancer (24), human prostate cancer (25) and murine models of lung tumourigenesis (23).
  • COX-1 expression can be induced in vitro by tobacco carcinogen (42), VEGF (21), arachidonic acid, forskolin, dibutryl-cAMP and PGE2 (22).
  • tobacco carcinogen 42
  • VEGF 21
  • arachidonic acid forskolin
  • dibutryl-cAMP dibutryl-cAMP
  • PGE2 PGE2
  • This study demonstrates up-regulation of COX-1 expression in squamous cell carcinoma and adenocarcinoma of the human cervix as demonsfrated by real-time quantitative RT-PCR, Western blot analysis and immunohistochemistry.
  • the up-regulation of expression of COX-1 was associated with enhanced expression of COX-2.
  • the site of COX-1 expression localised to the neoplastic epithelial cells of all squamous cell carcinomas and adenocarcinomas investigated, demonsfrating a similar pattern of expression for COX-1 in cancer of the cervix as has been demonstrated for COX-2 (16, 18) and PGE2 (18).
  • COX-2 expression in HeLa cells was only maximally induced at 72 hrs. This suggests that PGE2 production detected at 72 hrs after COX-1 overexpression is enhanced by the activity of both COX enzymes.
  • COX-2 expression is up-regulated by PGE2 via the cAMP-dependent PGE2-receptors (22).
  • cAMP activity accompanies a concomitant increase in COX-2 synthesis suggesting that cAMP is the primary secondary messenger in regulating COX-2, presumably via the upstream cAMP response element (CRE) located on the COX-2 gene (46).
  • CRE upstream cAMP response element
  • cAMP activity was measured in HeLa cells following overexpression of COX-1 for 48 hrs and stimulation with exogenous PGE2- A significant fold increase in cAMP production was observed after 5 min of PGE2 stimulation in COX-1 induced compared with uninduced cells. This augmented cAMP response was abolished by growing cells in medium containing indomethacin.
  • Cancer cells produce a wide variety of factors that contribute to angiogenesis, including bFGF, VEGF, bFGF-binding protein and platelet- derived growth factor (PDGF) (34).
  • bFGF vascular endothelial growth factor
  • VEGF vascular endothelial growth factor
  • PDGF platelet- derived growth factor
  • Ang-1 is a Tie-2 receptor agonist, which is required for recruitment of perivascular cells leading to the formation and stabilisation of capillaries, vessel maturation and endothelial cell survival (49, 50).
  • Ang-1 and other angiogenic factors such as VEGF may act synergistically to increase vascular sprouting and branching (51, 52).
  • Ang-l/Tie-2 interaction enhances the mitogenic effect of VEGF on endothelial cell growth (53).
  • Ang-2 is a natural Tie-2 receptor antagonist, destabilising cell contacts and thus allowing access to angiogenic factors such as VEGF (54).
  • VEGF vascular endothelial growth factor
  • enhanced synthesis of prostanoids as a consequence of upregulated COX-1 may thus act in an autocrine/paracrine manner to up- regulate the expression of COX-2 and target receptors as well as the intracellular signalling to a host of angiogenic factors, which could act on endothelial cells and lead to the recruitment of new blood vessels to enhance tumour mass.
  • Example 2 Cyclooxygenase-2 expression and prostaglandin E2 synthesis are up-regulated in carcinomas of the cervix; a possible autocrine/paracrine regulation of neoplastic cell function via EP2/EP4 receptors
  • Immunoreactive COX-2 and PGE2 were also co-localised to endothelial cells lining the microvasculature.
  • PGE2 has an autocrine/paracrine effect in cervical carcinomas
  • Cancer of the uterine cervix is one of the leading causes of cancer-related death in women world-wide. It is reported as being particularly common in less developed countries, including South and Central America, Southeast Asia and Sub-Saharan Africa (1, 2, 3), where 80% of the world's cervical cancers occur (4).
  • Three histological categories of epithelial tumours of the cervix are recognised by the World Health Organisation (5). These are squamous cell carcinoma, adenocarcinoma and other less common types of epithelial tumours.
  • the most common histological type of cervical carcinoma is squamous cell carcinoma that accounts for 60%- 80% of all cervical cancers. Adenocarcinoma accounts for approximately 20% of invasive cervical carcinoma.
  • Cyclooxygenase (COX) enzymes also called prostaglandin endoperoxide synthase, catalyse the rate limiting step in the conversion of arachidonic acid to prostaglandin H2 and other eicosanoids including prostaglandin E (6).
  • COX-1 and COX- 2 There are at least two isoforms of the COX enzyme, COX-1 and COX- 2 (7, 8).
  • COX-1 is constitutively expressed in many tissues and cell types and generates prostaglandins for normal physiological function (8).
  • the expression of COX-2 is rapidly induced following stimulation of quiescent cells by growth factors, oncogenes, carcinogens and tumour- promoting phorbal esters (7, 8, 9).
  • PGE2 Prostaglandin E2 elicits its autocrine/paracrine effects on target cells through interaction with seven transmembrane G-protein-coupled-receptors (GPCR) which belong to the rhodopsin family of serpentine receptors (10).
  • GPCR G-protein-coupled-receptors
  • COX-2 has been established (12, 13). Transcription of COX-2 is upregulated in numerous cancers including colon, pancreas, oesophagus, lung, prostate and bladder (14, 15, 16, 17, 18, 19). It has been proposed that COX-2 overexpression and PGE2 synthesis mediate neoplastic transformation of epithelial cells by increasing their proliferation rate, resistance to apoptosis and invasiveness. These effects are mediated by suppressing the transcription of target genes that may be involved in cellular growth/transformation (eg. p53) and adhesion (eg. E- Cadherin) (13, 20). Moreover, COX-2 and PGE2 promote cancer development and invasiveness by mediating the transcription of angiogenic factors that promote both migration of endothelial cells and their arrangements into tubular structures (21, 22).
  • target genes eg. p53
  • adhesion eg. E- Cadherin
  • the present study was designed to investigate whether COX-2 expression and PGE2 synthesis are upregulated in human squamous cell carcinomas and adenocarcinomas of the cervix.
  • a possible autocrine/paracrine role for PGE2 in cervical carcinogenesis was assessed by investigating (a) the expression of EP2/EP4 receptors in cervical carcinoma tissue and (b) the effect of exogenous freatment of carcinoma tissue with PGE2 on cAMP turnover.
  • Cervical specimens were obtained at the time of surgery/biopsy from patients that were attending the Gynaecologic Oncology Clinic at Groote Schuur Hospital, Cape Town and that had previously been diagnosed with invasive carcinoma of the cervix.
  • Punch biopsies were taken from the lesion by an experienced Gynaecologist with a special interest in oncology. A portion of the biopsy was excised and fixed in formalin followed by paraffin wax-embedding for histopathological typing. The remaining portion was snap frozen in either dry ice or liquid nitrogen and stored at -70°C for RT- PCR and Western blot analysis or transported at 4°C for in vitro culture and PGE2 stimulation. Histologically normal cervical samples were obtained from patients undergoing surgery for non-malignant conditions.
  • RNA samples were reverse transcribed using MgCl 2 (5.5 mM), dNTPs (0.5 mM each), random hexamers (1.25 ⁇ M), oligo-dT (1.25 ⁇ M), RNAase inhibitor (0.4 U/ ⁇ l) and multiscribe reverse transcriptase (1.25 U/ ⁇ l; all from PE Biosystems, Warrington, UK).
  • the mix was aliquoted into individual tubes (16 ⁇ l/rube) and template RNA was added (4 ⁇ l/tube of 100 ng/ ⁇ l RNA). Samples were incubated for 60 minutes at 25°C, 45 minutes at 48°C and then at 95°C for 5 minutes.
  • a reaction mix was made containing Taqman buffer (5.5 mM MgCl 2 , 200 ⁇ M dATP, 200 ⁇ M dCTP, 200 ⁇ M dGTP, 400 ⁇ M dUTP), ribosomal 18S forward and reverse primers and probe (all at 50 nM), forward and reverse primers for COX-2, EP2 or EP4 receptor (300 nM), COX-2, EP2 or EP4 receptor probe (200 nM), AmpErase UNG (0.01 U/ ⁇ l) and AmpHTaq Gold DNA Polymerase (0.025 U/ ⁇ l; all from PE Biosystems).
  • Taqman buffer 5.5 mM MgCl 2 , 200 ⁇ M dATP, 200 ⁇ M dCTP, 200 ⁇ M dGTP, 400 ⁇ M dUTP
  • ribosomal 18S forward and reverse primers and probe all at 50 nM
  • forward and reverse primers for COX-2, EP2 or EP4 receptor 300 n
  • a volume of 48 ⁇ l of reaction mix was aliquoted into separate tubes for each cDNA sample and 2 ⁇ l/replicate of cDNA was added. After mixing 23 ⁇ l of sample were added to the wells on a PCR plate. Each sample was added in duplicate. A no template control (containing water) was included in triplicate. Wells were sealed with optical caps and the PCR reaction run on an ABI Prism 7700 using standard conditions. COX-2 and EP receptor primers and probe for quantitative PCR were designed using the PRIMER express program (PE Biosystems).
  • the sequence of the COX-2 primers and probe were as follows: Forward: 5'-CCT TCC TCC TGT GCC TGA TG- 3'; Reverse: 5'- ACA ATC TCA TTT GAA TCA GGA AGC T-3'; Probe (FAM labelled): 5'- TGC CCG ACT CCC TTG GGT GTC A -3'.
  • the sequence of the EP2 receptor primers and probe were as follows; Forward: 5'-GAC CGC TTA CCT GCA GCT GTA C-3'; Reverse: 5'-TGA AGT TGC AGG CGA GCA-3'; Probe (FAM labelled): 5'-CCA CCC TGC TGC TGC TTC TCA TTG TCT- 3'.
  • the sequence of the EP4 receptor primers and probe were as follows; Forward: 5'-ACG CCG CCT ACT CCT ACA TG-3'; Reverse: 5'- AGA GGA CGG TGG CGA GAA T-3'; Probe (FAM labelled): 5'-ACG CGG GCT TCA GCT CCT TCC T-3'.
  • the ribosomal 18S primers and probe sequences were as follows; Forward: 5' -CGG CTA CCA CAT CCA AGG AA-3'; Reverse: 5'-GCT GGA ATT ACC GCG GCT-3'; Probe (VIC labelled): 5 -TGC TGG CAC CAG ACT TGC CCT C-3'.
  • sample buffer 125 mM Tris-HCl pH6.8, 4% SDS, 5% 2-mercaptoethanol, 20% glycerol and 0.05% bromophenol blue
  • Proteins were transferred onto polyvinyhdene difluoride membrane (PVDF, Millipore, Watford, UK) and subjected to immunoblot analysis. Membranes were blocked for 1 hour at 25°C in 5% skimmed milk powder diluted in washing buffer (50 mM Tris-HCl, 150 mM NaCl and 0.05% v/v Tween-20). Thereafter, membranes were incubated with goat anti-COX-2 primary IgG antibody (sc-1745; Autogenbioclear, Wiltshire, UK) at a dilution of 1:500 at 4°C for 18 hours.
  • PVDF polyvinyhdene difluoride membrane
  • Five-micron paraffin wax-embedded tissue sections were cut and mounted onto coated slides (TESPA, Sigma). Sections were dewaxed in xylene, rehydrated in graded ethanol and washed in water followed by TBS (50 mM Tris-HCl, 150 mM NaCl pH7.4) and blocked for endogenous endoperoxidase (1% H2O2 in methanol).
  • Antigen retrieval was performed by pressure cooking for 2 minutes in 0.1 M sodium citrate pH6 (for COX-2 and PGE2). No antigen retrieval was performed for CD34 immunohistochemistry. Sections were blocked using either 5% normal rabbit serum (for COX-2), 5% swine serum (for PGE2) or 5% normal goat serum (for CD34) diluted in TBS.
  • tissue sections were incubated with polyclonal goat anti-COX-2 antibody (sc-1745; Autogenbioclear) at a dilution of 1 :400, rabbit anti-PGE2 antibody (kindly supplied by Professor RW Kelly, MRC Human Reproductive Sciences Unit, Edinburgh, UK) at a dilution of 1:100 or monoclonal mouse anti-human CD34 primary antibody (mca-547; Serotec, Oxford, UK) at a dilution of 1 :25 at 4°C for 18 hours.
  • Control tissue was incubated with either 5% antisera (PGE2 and CD34) or goat anti-COX-2 antibody pre-adsorbed to blocking peptide (sc-1745p; Autogenbioclear).
  • tissue sections probed with the goat anti-human COX-2 and rabbit anti-PGE2 primary antibodies were incubated with biotinylated rabbit anti- goat secondary IgG antibody (for COX-2; Dako) or swine anti-rabbit secondary IgG antibody (for PGE2; Dako) at a dilution of 1 :500 at 25°C for 40 minutes. Thereafter the tissue sections were incubated with sfreptavadin- biotin peroxidase complex (Dako) at 25°C for 20 minutes. Tissue sections probed with the mouse anti-human CD34 antibody were developed using a Mouse En Vision Kit (Dako) as per the manufacturers instructions.
  • Dako Mouse En Vision Kit
  • DMEM Dulbecco's Modified Eagle Medium
  • tissue was snap frozen to determine basal cAMP concentration in the tissue at the time of collection.
  • the other two aliquots were incubated overnight at 37°C in humidified 5% C ⁇ 2 in the presence or absence of 3 ⁇ g/ml indomethacin [a dual COX enzyme inhibitor (24, 32)].
  • tissue sections were harvested by centrifugation at 2000 g. The supernatant was discarded and the tissue homogenised in 0.1 M HCl.
  • cAMP concentration was quantified by ELISA using a cAMP kit (Biomol; Affiniti, Singer, UK) as per the manufacturer's protocol and normalised to protein concentration of the homogenate. Protein concentrations were determined using protein assay kits (Bio-Rad, Hemel Hempstead, UK).
  • DMEM Dulbecco 's Modified Eagle Medium
  • samples were incubated in the same medium containing IBMX (Sigma) to a final concentration of 1 mM for 30 minutes at 37°C and then stimulated with 0 nM PGE2, 300 nM PGE2 or 50 ⁇ M forskolin (forskolin treatment in sample C2 was excluded due to the small size of the biopsy) for 5 minutes.
  • Tissue sections were harvested by centrifugation at 2000 g. The supernatant was discarded and the tissue homogenised in 0.1 M HCl.
  • cAMP concentration and protein concentrations were determined as mentioned above
  • COX-2 and PGE2 were upregulated in all carcinoma samples.
  • COX-2 and PGE2 were localised to the neoplastically transformed squamous epithelium in squamous cell carcinoma ( Figures 9a and 9b respectively), and to neoplastically transformed columnar epithelium lining the endocervical canal and the glandular epithelium of the endocervical glands in adenocarcinomas ( Figure 9c and 9d respectively).
  • cAMP concentration immediately after tissue excision was significantly higher in carcinoma compared with normal cervix (77.9 + 30.9 vs 32.5 ⁇ 8.7 pmol cAMP/mg protein; P ⁇ 0.05).
  • cAMP concentrations in carcinoma tissue following overnight incubation in the absence of indomethacin was similar to that detected in the tissue at the time of excision (64.2 ⁇ 5.1 pmol cAMP/mg protein) but was significantly reduced when the tissue was cultured in the presence of indomethacin (2.59 ⁇ 0.64 pmol cAMP/mg protein).
  • COX-2 is the predominant prostaglandin synthesised from arachidonic acid by COX-2 (25). Enhanced synthesis of PGE2 resulting from upregulated COX-2 could induce malignant change in epithelial cells through immunosuppression (6), inhibiting apoptosis (20), increasing metastatic potential of epithelial cells (26) and promoting angiogenesis (21, 22). COX-2 and PGE2 control the process of angiogenesis in tumours either directly or indirectly. In an in vitro model, overexpression of COX-2 and PGE2 in colon epithelial cells enhances the expression of angiogenic factors that act on endothelial cells resulting in enhanced cell migration and microvascular tube formation (21).
  • COX-2 and PGE2 produced by endothelial cells may also directly regulate the process of angiogenesis (22).
  • the arrangement of rat aortic endothelial cells into tubular structures is reduced following treatment with selective COX-2 inhibitors and this effect is partially reversed by co-treatment with PGE2 (22).
  • PGE2 PGE2
  • PGE2 acts on target cells through interaction with G-protein coupled receptors. To-date several of these receptors have been cloned which utilise alternate intracellular signalling pathways. In this study we investigated the expression of two PGE2 membrane-bound receptors, namely EP2 and EP4, which mediate their effect on target cells via the PKA pathway by activating adenylate cyclase and increasing intracellular cAMP (10). In cervical carcinomas, expression of EP2 and EP4 receptors is up-regulated compared with normal cervix. In addition, the basal cAMP concenfration in the carcinoma tissue is elevated compared with normal cervix. Treatment of the cervical tissue with the COX enzyme inhibitor indomethacin significantly reduced cAMP concenfration.
  • COX-2 inhibitors exhibit dramatic antineoplastic activity in a number of tumour model systems investigated thus far, including: colon cancer cells implanted into nude mice, tumour production in APC mutant mice and carcinogen-induced tumours in rats (27, 28, 29). This is mediated partially by reducing PGE2 synthesis in the COX-2 over-expressing cells which in turn down-regulates the survival, metastatic and angiogenic potential of the cancerous tissue (20, 21, 26). This has prompted the suggestion that the inhibition of PGE2 secretion by the application of COX-2 inhibitors may have an effect on growth and invasiveness of various carcinomas (21, 22, 26, 27). Such treatments may also be of benefit in regulating the growth of cervical carcinoma.
  • Example 3 Up-regulation of expression of cyclooxygenase-2 and prostaglandin E receptors (EP2 and EP4) in HeLa cells by seminal plasma
  • COX-2, EP2 and EP4 were measured by real-time RT-PCR.
  • Treatment of HeLa cells with seminal plasma for 24hrs resulted in up-regulation of expression of COX-2, EP2 and EP4 receptors (fold induction above basal was 20.2 ⁇ 9.26, 12.5 ⁇ 6.2 and 7.8 ⁇ 2.8 respectively, p less than 0.05).
  • Co-treatment of cells with the dual COX- enzyme inhibitor indomethacin abolished the up-regulation in EP2/EP4 receptor expression.
  • Treatment of cells with the selective COX-2 inhibitor NS-398, and the PKA inhibitor H-89 partially abolished the up-regulated receptor expression.
  • a patient, diagnosed with invasive cervical carcinoma is administered ibuprofen.
  • Example 5 Treating a neoplastic condition of the cervix with an EP4 receptor antagonist
  • a patient diagnosed with CIN III is administered 5-butyl-2,4-dihydro-4- [[2'-[N-(3-methyl-2-thiophenecarbonyl)sulfamoyl]bi ⁇ henyl-4-yl]methyl]-2- ⁇ 2-(trifluoromethyl)phenyl]- 1 ,2,4-triazol-3-one.
  • Example 6 Treating cervical cancer with a combination of COX-1 inhibitor and EP2 receptor antagonist
  • a patient diagnosed with cervical carcinoma in situ is administered the combination of AH23848B and sulindac.
  • Example 7 Suppository mg/suppository AH22921 (63 ⁇ m)* 250
  • the antagonist AH22921 is used as a powder wherein at least 90% of the particles are of 63 ⁇ m diameter or less.
  • Witepsol HI 5 is melted in a steam-jacketed pan at 45°C maximum.
  • the active ingredient is sifted through a 200 ⁇ m sieve and added to the molten base with mixing, using a silverson fitted with a cutting head, until a smooth dispersion is achieved. Maintaining the mixture at 45 °C, the remaining Witepsol HI 5 is added to the suspension and stirred to ensure a homogenous mix.
  • the entire suspension is passed through a 250 ⁇ m stainless steel screen and, with continuous stirring, is allowed to cool to 40° C. At a temperature of 38 ° C to 40°C 2.02 g of the mixture is filled into suitable plastic moulds. The suppositories are allowed to cool to room temperature.
  • An intrauterine device containing AH6809 is produced using standard technology.
  • the device is for use with women diagnosed with CIN I, II or III.
  • a tampon for halting the progression from CIN II to CIN III is produced by impregnating a standard tampon with an effective dose of 5-butyl-2,4- dihy dro-4- [ [2 '- [N- [2-(methypyrrole)carbonyl] sulfamoyl]biphenyl-4- yl]mefhyl]-2- ⁇ 2-(trifluoromethyl)phenyl]- 1 ,2,4-triazol-3-one.
  • the EP2 or EP4 receptor antagonist of Examples 7 to 11 may be used in combination with a COX-1 inhibitor, or may be replaced with another EP2 or EP4 receptor anatgonist or with a COX-1 inhibitor.
  • References for Example 1
  • Cyclooxygenase-2 is overexpressed in human cervical cancer. Clin Cancer Res, 7: 429-434., 2001.
  • Tsujii M DuBois RN. 1995 Alterations in cellular adhesion and apoptosis in epithelial cells overexpressing prostaglandin endoperoxide synthase 2. Cell. 83:493-501. 21. Tsujii M, Kawano S, Tsuji S, Sawaoka H, Hori M, DuBois RN. 1998 Cyclooxygenase regulates angiogenesis induced by colon cancer cells. Cell. 93:705-716. 22. Jones MK, Wang H, Peskar BM, et al. 1999 Inhibition of angiogenesis by nonsteroidal anti-inflammatory drags: insight into mechanisms and implications for cancer growth and ulcer healing. Nat Med. 5:1418-1423.

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Abstract

L'invention concerne une méthode de traitement d'un état néoplasique du col de l'utérus chez une patiente, qui consiste à administrer à la patiente un inhibiteur de la cyclooxygénase-1 (COX-1) et/ou un antagoniste du récepteur EP2 et/ou EP4.
PCT/GB2001/004826 2001-10-31 2001-10-31 Utiilisation d'un antagoniste du recepteur ep2 ou ep4 et/ou d'un inhibiteur de la cox-1 pour traiter un cancer cervical WO2003037373A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010087425A1 (fr) 2009-01-30 2010-08-05 国立大学法人京都大学 Inhibiteur de la progression du cancer de la prostate et procédé d'inhibition de la progression

Citations (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS53127832A (en) * 1977-04-11 1978-11-08 Teijin Ltd Antiphkogistics for oral administration containing thiaprostaglandins
EP0112633A2 (fr) * 1982-12-20 1984-07-04 Teijin Limited 5-Thia prostaglandines, leur préparation et application pharmaceutique
US5639738A (en) * 1992-02-20 1997-06-17 Hyal Pharmaceutical Corporation Treatment of basal cell carcinoma and actinic keratosis employing hyaluronic acid and NSAIDs
US5792753A (en) * 1991-07-03 1998-08-11 Hyal Pharmaceutical Corporation Compositions comprising hyaluronic acid and prostaglandin-synthesis-inhibiting drugs
WO2000018744A1 (fr) * 1998-09-25 2000-04-06 Fujisawa Pharmaceutical Co., Ltd. Composes d'oxazole convenant comme agonistes ou antagonistes de la prostaglandine e2
WO2000071135A1 (fr) * 1999-05-25 2000-11-30 Point Therapeutics, Inc. Agents anti-tumorales contenant des composes de boroproline
WO2001015730A1 (fr) * 1999-08-27 2001-03-08 Genentech, Inc. DOSAGES POUR TRAITEMENT AVEC DES ANTICORPS ANTI-ErbB2
EP1097922A1 (fr) * 1998-07-15 2001-05-09 Ono Pharmaceutical Co., Ltd. Derives de phenyl-postaglandine e 5-thia-omege-substitues, procede de production desdits derives et medicaments contenant lesdits derives en tant que principe actif

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS53127832A (en) * 1977-04-11 1978-11-08 Teijin Ltd Antiphkogistics for oral administration containing thiaprostaglandins
EP0112633A2 (fr) * 1982-12-20 1984-07-04 Teijin Limited 5-Thia prostaglandines, leur préparation et application pharmaceutique
US5792753A (en) * 1991-07-03 1998-08-11 Hyal Pharmaceutical Corporation Compositions comprising hyaluronic acid and prostaglandin-synthesis-inhibiting drugs
US5639738A (en) * 1992-02-20 1997-06-17 Hyal Pharmaceutical Corporation Treatment of basal cell carcinoma and actinic keratosis employing hyaluronic acid and NSAIDs
US5914322A (en) * 1992-02-20 1999-06-22 Hyal Pharmaceutical Corporation Treatment of disease and conditions
EP1097922A1 (fr) * 1998-07-15 2001-05-09 Ono Pharmaceutical Co., Ltd. Derives de phenyl-postaglandine e 5-thia-omege-substitues, procede de production desdits derives et medicaments contenant lesdits derives en tant que principe actif
WO2000018744A1 (fr) * 1998-09-25 2000-04-06 Fujisawa Pharmaceutical Co., Ltd. Composes d'oxazole convenant comme agonistes ou antagonistes de la prostaglandine e2
WO2000071135A1 (fr) * 1999-05-25 2000-11-30 Point Therapeutics, Inc. Agents anti-tumorales contenant des composes de boroproline
WO2001015730A1 (fr) * 1999-08-27 2001-03-08 Genentech, Inc. DOSAGES POUR TRAITEMENT AVEC DES ANTICORPS ANTI-ErbB2

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
PATENT ABSTRACTS OF JAPAN vol. 003, no. 003 (C - 033) 16 January 1979 (1979-01-16) *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2010087425A1 (fr) 2009-01-30 2010-08-05 国立大学法人京都大学 Inhibiteur de la progression du cancer de la prostate et procédé d'inhibition de la progression

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