Disclosed are compositions and a method for of amplifying nucleic acid sequences useful for detecting the presence of molecules of interest. The method is useful for detecting specific nucleic acids in a sample with high specificity and sensitivity. The method also has an inherently low level of background signal. A preferred form of the method consists of a DNA ligation operation, an amplification operation, and a detection operation. The DNA ligation operation circularizes a specially designed nucleic acid probe molecule. This operation is dependent on hybridization of the probe to a target sequence and forms circular probe molecules in proportion to the amount of target sequence present in a sample. The amplification operation is rolling circle replication of the circularized probe. A single round of amplification using rolling circle replication results in a large amplification of the circularized probe sequences. Following rolling circle replication, the amplified probe sequences are detected and quantified using any of the conventional detection systems for nucleic acids such as detection of fluorescent labels, enzyme-linked detection systems, antibody-mediated label detection, and detection of radioactive labels. Because, the amplified product is directly proportional to the amount of target sequence present in a sample, quantitative measurements reliably represent the amount of a target sequence in a sample. Major advantages of this method are that the ligation step can be manipulated to obtain allelic discrimination, the DNA replication step is isothermal, and signals are strictly quantitative because the amplification reaction is linear and is catalyzed by a highly processive enzyme. In multiplex assays, the primer oligonucleotide used for the DNA polymerase reaction can be the same for all probes. Also described are modes of the method in which additional amplification is obtained using a cascade of strand displacement reactions.

Rolling circle replication reporter systems

The invention is directed to antibody drug conjugate compositions of matter useful for the diagnosis and treatment of tumors in mammals and to methods of using those compositions of matter for the same.

Compositions and methods for the diagnosis and treatment of tumor

Compounds, compositions and methods are provided for modulating the expression and function of small non-coding RNAs. The compositions comprise oligomeric compounds, targeted to small non-coding RNAs. Methods of using these compounds for modulation of small non-coding RNAs as well as downstream targets of these RNAs and for diagnosis and treatment of disease associated with small non-coding RNAs are also provided.

Oligomeric compounds and compositions for use in modulation of small non-coding RNAs

The present invention provides 6-modified bicyclic nucleoside analogs and oligomeric compounds comprising these nucleoside analogs. In preferred embodiments the nucleoside analogs have either (R) or (S)-chirality at the 6-position. These bicyclicnucleoside analogs are useful for enhancing properties of oligomeric compounds including nuclease resistance.

6-modified bicyclic nucleic acid analogs

Oligomeric compounds including oligoribonucleotides and oligoribonucleosides are provided that have subsequences of 2′-pentoribofuranosyl nucleosides that activate dsRNase. The oligoribonucleotides and oligoribonucleosides can include substituent groups for increasing binding affinity to complementary nucleic acid strand as well as substituent groups for increasing nuclease resistance. The oligomeric compounds are useful for diagnostics and other research purposes, for modulating the expression of a protein in organisms, and for the diagnosis, detection and treatment of other conditions susceptible to oligonucleotide therapeutics. Also included in the invention are mammalian ribonucleases, i.e., enzymes that degrade RNA, and substrates for such ribonucleases. Such a ribonuclease is referred to herein as a dsRNase, wherein “ds” indicates the RNase's specificity for certain double-stranded RNA substrates. The artificial substrates for the dsRNases described herein are useful in preparing affinity matrices for purifying mammalian ribonuclease as well as non-degradative RNA-binding proteins.

Oligoribonucleotides and ribonucleases for cleaving RNA

A dinucleotide analogue of formula ##STR1## where B1 and B2 are each independently a monovalent nucleoside base radical; R1 is R1 a or Z; R1 a, R2, R3 and R4 are each independently hydrogen, halogen or hydroxy; R5 is R5 a or Z; R6 is hydrogen or R6 a ; R7 is hydrogen, alkyl-N,N-dialkylphosphoramidyl or R7 a, R8 is R8 a or Z, or the indicated R7 O and R8 together denote an isopropylidenedioxy group; R5 a and R8 a are each independently hydrogen, halogen, hydroxy, --OR10, --OCOR10 or silyloxy substituted by three C1 -C15 hydrocarbyl groups; R6 a and R7 a are each independently a C1 -C10 aliphatic radical, a C6 -C15 aromatic radical, a C7 -C30 araliphatic radical, --COR11, --SO2 R11 or silyl substituted by three C1 -C15 hydrocarbyl groups; R9 is hydrogen, a C1 -C8 aliphatic radical, a C3 -C8 cycloaliphatic radical, a C6 -C15 aromatic radical, a C7 -C13 araliphatic radical, an alkali metal ion or an ammonium ion; R10 and R11 are each independently a C1 -C10 aliphatic radical, a C3 -C8 cycloaliphatic radical, a C6 -C15 aromatic radical or a C7 -C16 araliphatic radical; Rx and Ry are independently hydrogen, halogen, hydroxy, a C1 -C10 alkyl, C2 -C10 alkenyl, C3 -C8 cycloalkyl, C6 -C15 aryl, C7 -C16 aralkyl, C1 -C10 alkoxy, C2 -C10 alkenoxy, C6 -C10 aryloxy or C7 -C16 aralkyloxy group, which is substituted or unsubstituted, or --OCORz ; Rz is a substituted or unsubstituted C1 -C10 alkyl, C2 -C10 alkenyl, C3 -C8 cycloalkyl, C6 -C15 aryl or C7 -C16 aralkyl group; and Z is C6 -C10 aryloxythiocarbonyloxy, the C6 -C10 aryl group being substituted or unsubstituted.

Dinucleoside phosphinates and their pharmaceutical compositions

A compound of formula ##STR1## where R1 is hydrogen or a protecting group Q; R2 is hydrogen, a C1 -C8 aliphatic radical, a C6 -C15 aromatic radical, a C3 -C8 cycloaliphatic radical, a C7 -C13 araliphatic radical, an alkali metal ion or an ammonium ion; R3 and R4 are independently hydrogen, halogen or hydroxy; R5 is C6 -C10 aryloxythiocarbonyloxy, the C6 -C10 aryl group being unsubstituted or substituted, or R5 a ; R5 a is hydrogen, fluorine, chlorine, hydroxy, --OR8, --OCOR8 or silyloxy substituted by three C1 -C15 hydrocarbyl groups; R6 is hydrogen, a C1 -C10 aliphatic radical, a C6 -C15 aromatic radical, a C7 -C16 araliphatic radical, --COR9, --SO2 R9 or silyl substituted by three C1 -C15 hydrocarbyl groups; R7 is a monovalent nucleoside base radical, hydroxyl, --OR8 or --OCOR8, and R9 are independently a C1 -C10 aliphatic radical, a C3 -C8 cycloaliphatic radical, a C6 -C15 aromatic radical or a C7 -C16 araliphatic radical; or R5 and the indicated R6 O-together denote an isopropylidenedioxy group or R5 and R7 together denote an isopropylidenedioxy group, provided that when R5 and R7 together denote isopropylidenedioxy, R1 R2, R3 R4 and R6 are not all hydrogen. The disclosure further relates to a method of preparing the compounds of formula I by reacting a olefinic acetonide with a phosphinate compound and to the use of the compounds of formula I as pharmaceutical agents.

Nucleoside phosphinate compounds and compositions

Rationale: Enhancing non-CFTR (cystic fibrosis transmembrane conductance regulator)-mediated anion secretion is an attractive therapeutic approach for the treatment of cystic fibrosis (CF) and other mucoobstructive diseases.Objectives: To determine the effects of TMEM16A potentiation on epithelial fluid secretion and mucociliary clearance.Methods: The effects of a novel low-molecular-weight TMEM16A potentiator (ETX001) were evaluated in human cell and animal models of airway epithelial function and mucus transport.Measurements and Main Results: Potentiating the activity of TMEM16A with ETX001 increased the Ca2+-activated Cl- channel activity and anion secretion in human bronchial epithelial (HBE) cells from patients with CF without impacting calcium signaling. ETX001 rapidly increased fluid secretion and airway surface liquid height in CF-HBE cells under both static conditions and conditions designed to mimic the shear stress associated with tidal breathing. In ovine models of mucus clearance (tracheal mucus velocity and mucociliary clearance), inhaled ETX001 was able to accelerate clearance both when CFTR function was reduced by administration of a pharmacological blocker and when CFTR was fully functional.Conclusions: Enhancing the activity of TMEM16A increases epithelial fluid secretion and enhances mucus clearance independent of CFTR function. TMEM16A potentiation is a novel approach for the treatment of patients with CF and non-CF mucoobstructive diseases.

TMEM16A Potentiation: A Novel Therapeutic Approach for the Treatment of Cystic Fibrosis

A compound of formula (I) in free or salt form, where R1 is hydrogen or alkyl optionally substituted by hydroxy, alkoxy, or alkylthio, R2 is hydrogen, alkyl, hydroxyalkyl, alkylcarbonyloxyalkyl, alkoxyalkyl, alkylthioalkyl, alkenyl, cycloalkylalkyl, heterocyclylalkyl, aralkyl in which the aryl ring thereof is optionally fused to a 5-membered heterocyclic group or is optionally substituted by one or more substituents selected from alkoxy, amino, alkylamino, dialkylamino, acylamino, halogen, hydroxy, aminosulfonyl, alkylaminosulfonyl, dialkylaminosulfonyl, alkylsulfonylamino or dialkylaminosulfonylamino, R3 is hydrogen or alkyl optionally substituted by hydroxy, alkoxy, or alkylthio, R4 is hydrogen or alkyl, R5 is a quinolinyl, isoquinolinyl or oxodihydroisoquinolinyl group optionally fused to a 5-membered heterocyclic group and optionally substituted by one or more substituents selected from halogen, cyano, hydroxy, alkyl, hydroxyalkyl, alkoxyalkyl, alkylthioalkyl, alkoxy, alkylthio, alkenyl, alkoxycarbonyl, alkynyl, carboxyl, acyl, a group of formula -N(R6)R7, aryl optionally substituted by one or more substituents selected from halogen or alkoxy, or heteroaryl having 5 or 6 ring atoms attached through a ring carbon atom to the indicated carbon atom, and R6 and R7 are each independently hydrogen or alkyl optionally substituted by hydroxy or alkoxy or one of R6 and R7 is hydrogen and the other is acyl, or R6 and R7 together with the nitrogen atom to which they are attached denote a 5- or 6- membered heterocyclyl group.

8-Quinolinxanthine and 8-isoquinolinxanthine derivatives as PDE 5 inhibitors

A medicament comprising, separately or together (A) glycopyrrolate; and (B) either a compound of formula (I) in free or salt or solvate form, wherein W, Rx, Ry, R1, R2, R3, R4, R5, R6 and R7 have the meanings as indicated in the specification, or a compound of formula (II) in free or salt or solvate form, wherein X has the meaning as indicated in the specification, for simulataneous, sequential or separate administration in the treatment of an inflammatory or obstructive airways disease. Pharmaceutical compositions that contain (A) and (B) are also described.

Stephen Paul Collingwood

Papers

Dinucleoside phosphinates and their pharmaceutical compositions

Nucleoside phosphinate compounds and compositions

TMEM16A Potentiation: A Novel Therapeutic Approach for the Treatment of Cystic Fibrosis

8-Quinolinxanthine and 8-isoquinolinxanthine derivatives as PDE 5 inhibitors

Combinations of Glycopyrrolate and Beta2 Adrenoceptor Agonists