WO2000015845A1 - T-type calcium channel - Google Patents
T-type calcium channel Download PDFInfo
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- WO2000015845A1 WO2000015845A1 PCT/US1999/019675 US9919675W WO0015845A1 WO 2000015845 A1 WO2000015845 A1 WO 2000015845A1 US 9919675 W US9919675 W US 9919675W WO 0015845 A1 WO0015845 A1 WO 0015845A1
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- type calcium
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
Definitions
- the present invention relates generally to calcium channel proteins, and more particularly to pancreatic T- type calcium channel proteins and uses thereof.
- Insulin secretion from pancreatic ⁇ -cells is the primary physiological mechanism of blood glucose regulation. A rise in blood glucose concentration stimulates release of insulin from the pancreas, which in turn promotes glucose uptake in peripheral tissues and consequently lowers blood glucose levels, reestablishing euglycemia.
- NIDDM non-insulin dependent diabetes mellitus
- pancreatic ⁇ -cells Vague and Moulin, 1982
- Voltage-gated Ca 2+ channels mediate a rapidly activated inward movement of Ca 2+ ions that underlies the stimulation of insulin secretion m ⁇ -cells (Boyd III 1991) .
- four types of Ca 2 " channels have been described (L(P/Q), T, N, and E channels) .
- the purified L-type Ca 2+ channel consists of five subunits: ⁇ 1 ⁇ 2 ⁇ , ⁇ , ⁇ (Catterall 1991) .
- the primary structure of the a 1 subunit is organized m four homologous domains containing six transmembrane segments (Catterall 1988) .
- Rat and human pancreatic ⁇ -cells are equipped with L-type and T-type Ca 2 ⁇ channels (Hi ⁇ art and Matteson, 1988; Davalli et al . , 1996).
- L-type Ca 2+ channels activated at high voltages and having large unitary conductance and dihydropy ⁇ dme-sensitivity, are considered the manor pipeline for Ca 2+ influx into the ⁇ - cell (Keahey et al . , 1989) .
- T-type calcium channels activate at low voltages and have small unitary conductance and dihydropy ⁇ dme-msensitivity.
- T-type Ca 2 channels m ⁇ -cell msulm-secretion have been demonstrated (Bhattacharjee et al . , 1997) . These channels facilitate exocytosis by enhancing electrical activity m these cells.
- L-type and T-type Ca 2+ channels under normal conditions, work m concert promoting the rise m [Ca 2+ ] ⁇ during glucose-stimulated insulin secretion.
- over-expressed T-type Ca 2+ channels may be, at least m part, responsible for the hyper-responsiveness of insulin secretion to non-glucose depolarizing stimuli m GK rat and m rat with NIDDM induced by neonatal injection of streptozotocm (Kato et al . , 1994; Kato et al . , 1996).
- over-expressed T-type calcium channels over time will ultimately lead to an elevation of basal Ca 2+ through it's window current properties. Therefore, there is a dual effect of T-type Ca 2+ channels m ⁇ -cells depending upon channel number and membrane potential.
- T-type calcium channels are associated with type II diabetes, a need exists to further characterize T-type calcium channels.
- the subject invention provides an isolated nucleic acid molecule encoding a pancreatic T- type calcium channel.
- the invention also provides an antisense nucleic acid molecule complementary to at least a portion of the mRNA encoding the pancreatic T-type calcium channel .
- the isolated nucleic acid molecules of the invention can be inserted into suitable expression vectors and/or host cells. Expression of the nucleic acid molecules encoding the pancreatic T-type calcium channel results m production of pancreatic T-type calcium channel m a host cell . Expression of the antisense nucleic acid molecules m a host cell results m decreased expression of the pancreatic T-type calcium channel.
- the invention further provides a ribozyme having a recognition sequence complementary to a portion of mRNA encoding a pancreatic T-type calcium channel. The ribozyme can be introduced into a cell to also achieve decreased expression of pancreatic T-type calcium channel m the cell .
- the invention further provides a method of screening a substance for the ability of the substance to modify T- type calcium channel function, and a method of obtaining DNA encoding a pancreatic T-type calcium channel.
- an isolated nucleic acid molecule encoding a pancreatic T-type calcium channel, wherein the nucleic acid molecule encodes a first ammo acid sequence having at least 90% ammo acid identity to a second ammo acid sequence .
- the second ammo acid ⁇ sequence is as shown m SEQ ID NO : 2.
- the invention further provides a DNA oligomer capable of hybridizing to a nucleic acid molecule encoding a pancreatic T-type calcium channel.
- the DNA oligomer can be used m a method of detecting presence of a pancreatic T-type calcium channel m a sample, which method is also provided by the subject invention.
- the invention also provides an isolated pancreatic T-type calcium channel protein, and antibodies or antibody fragments specific for the pancreatic T-type calcium channel protein.
- the antibodies and antibody fragments can be used to detect the presence of the pancreatic T-type calcium channel protein m samples.
- an isolated pancreatic T-type calcium channel protein encoded by a first ammo acid sequence having at least 90% ammo acid identity to a second ammo acid sequence, the second ammo acid sequence as shown m SEQ ID NO: 2.
- the subject invention further provides a method of modifying insulin secretion by pancreatic beta cells, the method comprising modifying levels of functional T type calcium channels m the pancreatic beta cells.
- the invention further provides a method of treating type II diabetes m a subject, the method comprising administering to the subject an amount of a compound effective to modify levels of functional T type calcium channel m the pancreatic beta cells of the subject.
- the invention also provides a method of modifying basal calcium levels m cells, a method of modifying the action potential of L type calcium channels m cells, a method of modifying pancreatic beta cell death, a method of modifying pancreatic beta cell proliferation, and a method of modifying calcium influx through L type calcium channels m cells, each of the methods comprising modifying levels of functional T type calcium channels m the cells.
- Fig. IA illustrates a comparison of the nucleotide sequence of c ⁇ G-INS (1) and c ⁇ G (2) at the 5 ' -end regions (aal-67 of a x G) .
- the four insertions are indicated with arrow heads.
- the capital ATG represents the start codon for each cDNA;
- Fig. IB is a schematic illustration representing partial rat genomic nucleotide composition between Domain III and IV.
- Genomic DNA contained an exon specific to ⁇ x G (shaded circle) and an exon specific to the ⁇ 1 subunit of T-type Ca 2+ deduced from INS-1 (shaded rectangle) between 4845 and 5256 of the cDNA sequence. Other exons (open rectangles) are identical between the two cDNAs .
- the bold letters indicate the nucleotides coding Gly-1667;
- Figs. 2A-2D illustrate expression of o ⁇ G-INS m Xenopus oocytes .
- Fig. 2A illustrates 40 mM Ca 2+ currents elicited by depolarizing pulses from - 60 to 40 mV.
- the holding potential for Figs. 2C and 2D was -80 mV.
- the currents m Fig. 2D were measured at -10 mV after varying 1000 ms pre-pulse potentials. Peak currents were normalized to the maximum current and then averaged (error bars represent SE) ;
- Figs. 3A and 3B illustrate accumulative dose response relationships of the inhibitory effects of mibefradii on T- and L-type Ca 2+ currents. Currents were measured with the whole-cell patch clamp configuration. Data from four experiments were normalized individually and than plotted as mean ⁇ standard error.
- Fig. 3A illustrates curve which was generated by fitting the data using one-to-one binding curve according to the equation 1/(1 + [mibefradil] /K d ) .
- Fig. 3B is a dose response of L-type Ca 2+ current obtained when perfusion of solutions containing different concentrations of mibefradil;
- Fig. 4 illustrates reversibility of the inhibition of T and L-type currents by N ⁇ Cl 2 and mibefradil, respectively.
- Open and solid circles represent the T-type Ca 2+ current recorded before and after N ⁇ Cl 2 (2 ⁇ l of 30 ⁇ M) and mibefradil (2 ⁇ l of 10 ⁇ M) were administrated, respectively.
- the open squares represent the L-type Ca 2+ current recorded before and after mibefradil (2 ⁇ l of 10 ⁇ M) was administrated with perforated patch clamp configuration.
- FIG. 5A and 5B illustrate the long-term effect of mibefradil (10 nM) on L- and T- Ca 2+ currents m the perforated-patch configuration.
- solid and open circles represent the L-type Ca 2 current recorded m the cells with and without administration of mibefradil, " ⁇ respectively.
- Fig. 6A illustrates accumulation of dm-mibefradii m the cells measured with mass spectrometry.
- the inset (Fig. 6B) shows the primary data of mass spectrometry indicating peaks at 496 and 424, which correspond to mibefradil and dm-mibefradii , respectively;
- the pipette solution contained 1 ⁇ M of drug ;
- Fig. 8 illustrates basal [Ca 2+ ] . measured an INS-1 cell.
- T-type calcium channel antagonist mibefradil (1 ⁇ M) reduced basal [Ca 2+ ] . m a single cell m the bath solution without glucose.
- the [Ca 2+ ] . was measured with the emission ratio of Fura-2 AM (F380/F340) then calibrated with the standard solution purchased from Molecular Probes Inc. (OR);
- Fig. 9A illustrates that intracellular perfusion of a solution containing 272 nM free calcium concentration inhibits the L-type calcium current. Currents were elicited by a step voltage to +10 mV, with holding potential of -80 mV;
- Fig. 9B illustrates the effect of perfusing m high calcium concentration on the IV calcium current relationship. Closed circles represent the cell before perfusion, and open circles represent perfusion of 272 nM free calcium;
- Fig. 9C illustrates the effect of intracellular perfusion of different calcium concentrations on L-type calcium current over time.
- Squares represent perfusion from high calcium to low calcium (intracellular solution contained 632 nM then perfused by a solution with 10 mM EGTA)
- triangles represent perfusion from low calcium to 272 nM calcium
- circles represent low calcium to 632 nM calcium;
- Fig. 9D illustrates the effect of high calcium on the T-type calcium channel current. Tail currents were elicited by a voltage step to -30 mV for 10 ms ;
- Fig. 10 illustrates that reestablishment of basal calcium causes stereotyped calcium influx.
- a cell was twice perfused with 50 mM KCl with an intervening perfusion of the original bath solution to restore membrane potential;
- Fig. 11 illustrates that elevated basal Ca 2+ causes a defect m the Ca 2+ transient.
- a cell was twice perfused with 50 mM KCl with an intervening perfusion of the original bath solution to restore membrane potential . The second perfusion occurred prior to reestablishment of the original basal [Ca 2+ ] . of about 60 nM;
- Fig. 12 illustrates a model for glucose-stimulated insulin release;
- Fig. 13 illustrates that mibefradil (1 ⁇ M) blocks T- and L-type Ca 2+ current m INS-1 cells.
- Fig. 15 illustrates the activation and mactivation curves for INS-1 cells, revealing a "window current"
- Fig. 16 illustrates the effect of N ⁇ Cl 2 , mibefradil, and nifedipme on basal insulin secretion m NIT-1 cells.
- the glucose concentration is 3 mM m the experiments;
- Fig. 17 illustrates that the T type calcium channel antagonist N ⁇ Cl 2 (30 ⁇ M) reduced the frequency of transient spontaneous elevation of [Ca 2 ⁇ , m a single cell m the bath solution without glucose;
- Figs. 20A and 20B illustrate the dose-dependent effect of N ⁇ Cl 2 on insulin secretion.
- Fig. 21 illustrates "run-up" m whole cell recording
- Fig. 22 illustrates KCl induced Ca 2+ influx m the INS-1 cells treated with streptozotocm.
- n 13;
- Fig. 23A-23F illustrate the results of cytokme treatment .
- LVA Ca + currents were induced by cytokme treatment (IL-l ⁇ , 25 U/ml ; IFN ⁇ , 300 U/ml) for 6 h m primary cultured mouse islet cells, but not ⁇ -TCl cells.
- An LVA current was elicited by a -40 mV test — pulse m an islet cell (Fig. 23A) , but the same current was not detected m ⁇ -TCl cells (Fig. 23C) .
- the Ca 2+ current density-voltage relationships obtained from islet cells (Fig. 23B) and ⁇ -TCl cells (Fig. 23D) with and without cytokme treatment are shown.
- the recordings were elicited by voltages ranging from -50 to +20 mV for 100 msec. All experiments were performed at -80 mV.
- FIG. 23E shows steady state mactivation of LVA tail currents elicited by a 10-msec depolarizing (-10 mV) pulse followed by a 50-msec hyperpolarizmg pulse (-100 mV) , with a holding potential of -80 mV.
- Fig. 23F shows that N ⁇ Cl 2 (10 ⁇ M) blocked the cytokme induced LVA Ca 2+ current elicited at a -30 mV step pulse m an islet cell;
- Figs. 24A and 24B illustrate the effects of cytokmes on [Ca 2+ ] m mouse islet cells and ⁇ -TCl cells.
- basal [Ca 2+ ] of primary cultured mouse islet cells was approximately 3 -fold higher after cytokme treatment.
- N ⁇ Cl 2 (10 ⁇ M) but not mfedipme (10 ⁇ M) , prevented the increase m [Ca 2"1" ].
- m ⁇ -TCl cells was unaffected by cytokme treatment. Cytokme treatment consisted of IL-l ⁇ (25 U/ml) and IFNy (300 U/ml) for 6 h; and
- Figs. 25A and 25B illustrate the effects of N ⁇ Cl 2 on cytokme-induced ⁇ -TC3 cell death.
- Cytokine treatment consisted of IL-l ⁇ (25 U/ml), IFN ⁇ (100 U/ml), and TNF ⁇ (100 U/ml) m
- the first dose, 0, represents zero concentration for all three cytokmes.
- the concentration of nifedipine was 10 ⁇ M in both Fig. 25A and Fig. 25B.
- nucleic acid refers to either DNA or RNA.
- Nucleic acid sequence or “polynucleotide sequence” refers to a single- or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. It includes both self-replicating plasmids, infectious polymers of DNA or RNA, and nonfunctional DNA or RNA.
- isolated nucleic acid refers to nucleic acid which has been separated from an organism in a substantially purified form (i.e. substantially free of other substances originating from that organism) , and to synthetic nucleic acid.
- nucleic acid sequence “homologous to” or “complementary to” it is meant a nucleic acid that selectively hybridizes, duplexes or binds to DNA sequences encoding the protein (channel) or portions thereof when the DNA sequences encoding the protein are present in a human genomic or cDNA library.
- a DNA sequence which is similar or complementary to a target sequence can include sequences which are shorter or longer than the target sequence so long as they meet the functional test set forth.
- the hybridization is done m a Southern blot protocol using a 0.2X SSC, 0.1% SDS, 65°C wash.
- SSC refers to a citrate-salme solution of 0.15M sodium chloride and 20 mM sodium citrate.
- 6X SSC refers to a solution having a sodium chloride and sodium citrate concentration of 6 times this amount or 0.9 M sodium chloride and 120 mM sodium citrate.
- 0.2X SSC refers to a solution 0.2 times the SSC concentration or 0.03M sodium chloride and 4 mM sodium citrate.
- nucleic acid molecule encoding refers to a nucleic acid molecule which directs the expression of a specific protein or peptide.
- the nucleic acid sequences include both the DNA strand sequence that is transcribed into RNA and the RNA sequence that is translated into protein or peptide.
- the nucleic acid molecule includes both the full length nucleic acid sequences as well as non-full length sequences derived from the full length protein. It being further understood that the sequence includes the degenerate codons of the native sequence or sequences which may be introduced to provide codon preference m a specific host cell.
- located upstream refers to linkage of a promoter upstream from a nucleic acid (DNA) sequence such that the promoter mediates transcription of the nucleic acid (DNA) sequence.
- vector refers to viral expression systems, autonomous self-replicating circular DNA (plasmids) , and includes both expression and nonexpression plasmids. Where a recombinant microorganism or cell is described as hosting an "expression vector, " this includes both extrachromosomal circular DNA and DNA that has been incorporated into the host chromosome (s) . Where a vector is being maintained by a host cell, the vector may either be stably replicated by the cells during mitosis as an autonomous structure, or the vector may be incorporated within the — host ' s genome .
- plasmid refers to an autonomous circular DNA molecule capable of replication in a cell, and includes both the expression and nonexpression types . Where a recombinant microorganism or cell is described as hosting an "expression plasmid", this includes latent viral DNA integrated into the host chromosome (s) . Where a plasmid is being maintained by a host cell, the plasmid is either being stably replicated by the cell during mitosis as an autonomous structure, or the plasmid is incorporated within the host's genome.
- heterologous protein or “recombinantly produced heterologous protein” refers to a peptide or protein of interest produced using cells that do not have an endogenous copy of DNA able to express the peptide or protein of interest.
- the cells produce the peptide or protein because they have been genetically altered by the introduction of the appropriate nucleic acid sequences.
- the recombinant peptide or protein will not be found in association with peptides or proteins and other subcellular components normally associated with the cells producing the peptide or protein.
- reference sequence is a defined sequence used as a basis for a sequence comparison; a reference sequence may be a subset of a larger sequence, for example, as a segment of a full-length cDNA or gene sequence given m a sequence — listing or may comprise a complete cDNA or gene sequence.
- Optimal alignment of sequences for aligning a comparison window may be conducted, for example, by the local homology algorithm of Smith and Waterman (1981), by the homology alignment algorithm of Needleman and Wunsch (1970) , by the search for similarity method of Pearson and Lipman (1988), or by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA m the Wisconsin Genetics Software Package Release 7.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.) .
- nucleic acid molecules or polynucleotides As applied to nucleic acid molecules or polynucleotides, the terms “substantial identity” or “substantial sequence identity” mean that two nucleic acid sequences, when optimally aligned (see above), share at least 90 percent sequence identity, preferably at least 95 percent sequence identity, more preferably at least 96, 97, 98 or 99 percent sequence identity.
- Percentage nucleotide (or nucleic acid) identity or “percentage nucleotide (or nucleic acid) sequence identity” refers to a comparison of the nucleotides of two nucleic acid molecules which, when optimally aligned, have approximately the designated percentage of the same nucleotides.
- 95% nucleotide identity refers to a comparison of the nucleotides of two nucleic acid molecules which when optimally aligned have 95% nucleotide identity.
- nucleotide positions which are not identical differ by redundant nucleotide substitutions (the nucleotide substitution does not change the ammo acid encoded by the particular codon) .
- nucleic acid molecules or polynucleotides As further applied to nucleic acid molecules or polynucleotides , the terms “substantial homology” or “substantial sequence homology” mean that two nucleic acid sequences, when optimally aligned (see above), share at least 90 percent sequence homology, preferably at — least 95 percent sequence homology, more preferably at least 96, 97, 98 or 99 percent sequence homology.
- Percentage nucleotide (or nucleic acid) homology or “percentage nucleotide (or nucleic acid) sequence homology” refers to a comparison of the nucleotides of two nucleic acid molecules which, when optimally aligned, have approximately the designated percentage of the same nucleotides or nucleotides which are not identical but differ by redundant nucleotide substitutions (the nucleotide substitution does not change the ammo acid encoded by the particular codon) .
- “95% nucleotide homology” refers to a comparison of the nucleotides of two nucleic acid molecules which when optimally aligned have 95% nucleotide homology.
- the terms "substantial identity” or “substantial sequence identity” mean that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap, share at least 90 percent sequence identity, preferably at least 95 percent sequence identity, more preferably at least 96, 97, 98 or 99 percent sequence identity.
- Percentage ammo acid identity or “percentage ammo acid sequence identity” refers to a comparison of the ammo acids of two polypeptides which, when optimally aligned, have approximately the designated percentage of the same ammo acids.
- 95% ammo acid identity refers to a comparison of the ammo acids of two polypeptides which when optimally aligned have 95% ammo acid identity.
- residue positions which are not identical differ by conservative ammo acid substitutions.
- the substitution of ammo acids having similar chemical properties such as charge or polarity are not likely to affect the properties of a protein. Examples include glutamme for asparagme or — glutamic acid for aspartic acid.
- the terms "substantial homology” or “substantial sequence homology” mean that two peptide sequences, when optimally aligned, such as by the programs GAP or BESTFIT using default gap, share at least 90 percent sequence homology, preferably at least 95 percent sequence homology, more preferably at least 96, 97, 98 or 99 percent sequence homology.
- Percentage ammo acid homology or “percentage ammo acid sequence homology” refers to a comparison of the ammo acids of two polypeptides which, when optimally aligned, have approximately the designated percentage of the same ammo acids or conservatively substituted ammo acids.
- 95% ammo acid homology refers to a comparison of the ammo acids of two polypeptides which when optimally aligned have 95% ammo acid homology.
- homology refers to identical ammo acids or residue positions which are not identical but differ only by conservative ammo acid substitutions. For example, the substitution of ammo acids having similar chemical properties such as charge or polarity are not likely to affect the properties of a protein. Examples include glutamme for asparagme or glutamic acid for aspartic
- substantially purified or “isolated” when referring to a protein (or peptide) , means a chemical composition which is essentially free of other cellular components. It is preferably m a homogeneous state although it can be m either a dry or aqueous solution. Purity and homogeneity are typically determined using analytical chemistry techniques such as polyacrylamide gel electrophoresis or high performance liquid chromatography. A protein (or peptide) which is the predominant species present a preparation is — substantially purified. Generally, a substantially purified or isolated protein (or peptide) will comprise more than 80% of all macromolecular species present m the preparation.
- the protein (or peptide) is purified to represent greater than 90% of all macromolecular species present. More preferably the protein (or peptide) is purified to greater than 95%, and most preferably the protein (or peptide) is purified to essential homogeneity, wherein other macromolecular species are not detected by conventional techniques.
- a "substantially purified” or “isolated” protein (or peptide) can be separated from an organism, synthetically or chemically produced, or recombmantly produced.
- Biological sample or “sample” as used herein refers to any sample obtained from a living organism or from an organism that has died. Examples of biological samples include body fluids and tissue specimens.
- High stringent hybridization conditions are selected at about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH .
- Tm is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
- stringent conditions will be those m which the salt concentration is at least about 0.02 molar at pH 7 and the temperature is at least about 60°C.
- the combination of parameters is more important than the absolute measure of any one .
- High stringency may be attained, for example, by overnight hybridization at about 68°C m a 6X SSC solution, washing— at room temperature with 6X SSC solution, followed by washing at about 68°C m a 6X SSC solution then m a 0.6X SSX solution.
- Hybridization with moderate stringency may be attained, for example, by: 1) filter pre-hybridizmg and hybridizing with a solution of 3X sodium chloride, sodium citrate (SSC), 50% formamide, 0.1M Tris buffer at pH 7.5, 5X Denhardt ' s solution; 2) pre-hybridization at 37°C for 4 hours; 3) hybridization at 37°C with amount of labeled probe equal to 3,000,000 cpm total for 16 hours; 4) wash m 2X SSC and 0.1% SDS solution; 5) wash 4X for 1 minute each at room temperature and 4X at 60 °C for 30 minutes each; and 6) dry and expose to film.
- SSC sodium citrate
- selectively hybridizing to refers to a nucleic acid molecule that hybridizes, duplexes or binds only to a particular target DNA or RNA sequence when the target sequences are present a preparation of total cellular DNA or RNA.
- selectively hybridizing meant that a nucleic acid molecule binds to a given target m a manner that is detectable m a different manner from non-target sequence under moderate, or more preferably under high, stringency conditions of hybridization.
- “Complementary" or “target” nucleic acid sequences refer to those nucleic acid sequences which selectively hybridize to a nucleic acid molecule.
- Proper annealing conditions depend, for example, upon a nucleic acid molecule's length, base composition, and the number of mismatches and their position on the molecule, and must often be determined empirically.
- nucleic acid molecule (probe) design and annealing conditions see, for example, Sambrook et al . 1989.
- the DNA molecules of the subject invention also include DNA molecules coding for protein analogs, fragments or derivatives of the protein which differ from naturally-occurring forms (the naturally-occurring protein) m terms of the identity or location of one or more ammo acid residues (deletion analogs containing less than all of the residues specified for the protein, substitution analogs wherein one or more residues specified are replaced by other residues, and addition analogs wherein one or more ammo acid residues is added to a terminal or medial portion of the protein) and which share the signal property of the naturally-occurring form.
- These molecules include: the incorporation of codons "preferred" for expression by selected non-mammalian hosts; the provision of sites for cleavage by restriction endonuclease enzymes; and the provision of additional initial, terminal or intermediate DNA sequences that facilitate construction of readily expressed vectors .
- a "peptide” refers to an ammo acid sequence of three to one hundred ammo acids, and therefore an isolated peptide that comprises an amino acid sequence is not intended to cover amino acid sequences of greater than 100 amino acids.
- the peptides that can be identified and used in accordance with the subject invention are less than 50 amino acids in length, and more preferably the peptides are five to 20 amino acids in length or 20-40 amino acids in length.
- the peptides can contain any naturally-occurring or non-naturally-occurring amino acids, including the D-form of the amino acids, amino acid derivatives and amino acid mimics, so long as the desired function and activity of the peptide is maintained.
- the choice of including an (L) - or a (D) -amino acid in the peptides depends, in part, on the desired characteristics of the peptide. For example, the incorporation of one or more (D) -amino acids can confer increased stability on the peptide and can allow a peptide to remain active in the body for an extended period of time. The incorporation of one or more (D) -amino acids can also increase or decrease the pharmacological activity of the peptide.
- the peptides may also be cyclized, since cyclization may provide the peptides with superior properties over their linear counterparts.
- amino acid mimic and “mimetic” mean an amino acid analog or non-amino acid moiety that has the same or similar functional characteristic of a given amino acid.
- an amino acid mimic of a hydrophobic amino acid is one which is non-polar and retains hydrophobicity, generally by way of containing an aliphatic chemical group.
- an arginine mimic can be an analog of arginine which contains a side chain having a positive charge at physiological pH, as is characteristic of the guanidinium side chain reactive group of arginine.
- modifications to the peptide backbone and peptide bonds thereof are also encompassed within the scope of amino acid mimic or mimetic. Such modifications- can be made to the amino acid, derivative thereof, non-amino acid moiety or the peptide either before or after the amino acid, derivative thereof or non-amino acid moiety is incorporated into the peptide. What is critical is that such modifications mimic the peptide backbone and bonds which make up the same and have substantially the same spacial arrangement and distance as is typical for traditional peptide bonds and backbones.
- An example of one such modification is the reduction of the carbonyl (s) of the amide peptide backbone to an amine.
- An amino acid mimic is, therefor, an organic molecule that retains the similar amino acid pharmacophore groups as is present in the corresponding amino acid and which exhibits substantially the same spatial arrangement between functional groups.
- substitution of amino acids by non-naturally occurring amino acids and amino acid mimics as described above can enhance the overall activity or properties of an individual peptide based on the modifications to the backbone or side chain functionalities.
- these types of alterations to the specifically described amino acid substituents and exemplified peptides can enhance the peptide ' s stability to enzymatic breakdown and increase biological activity. Modifications to the peptide backbone similarly can add stability and enhance activity.
- SMPS simultaneous multiple peptide synthesis
- Peptides prepared by the method of Merrifield can be synthesized using an automated peptide synthesizer such as the Applied Biosystems 431A-01 Peptide Synthesizer (Mountain View, Calif.) or using the manual peptide synthesis technique described by Houghten, Proc. Natl. Acad. Sci., USA 82:5131 (1985).
- the subject invention provides an isolated nucleic acid molecule encoding a pancreatic T-type calcium channel.
- the nucleic acid molecule can be deoxyribonucleic acid (DNA) or ⁇ bonucleic acid (RNA, including messenger RNA or mRNA), genomic or recombinant, biologically isolated or synthetic .
- the DNA molecule can be a cDNA molecule, which is a DNA copy of a messenger RNA (mRNA) encoding the channel .
- mRNA messenger RNA
- An example of such a pancreatic T-type calcium channel is the rat pancreatic T-type calcium channel encoded by the nucleotide sequence as shown m SEQ ID NO : 1.
- the ammo acid sequence encoded by this nucleotide sequence is shown m SEQ ID NO : 2.
- the invention also provides an antisense nucleic acid molecule that is complementary to at least a portion of the mRNA encoding the pancreatic T-type calcium channel .
- Antisense nucleic acid molecules can be RNA or single- stranded DNA, and can be complementary to the — entire mRNA molecule encoding the channel (i.e. of the same nucleotide length as the entire molecule) . It may be desirable, however, to work with a shorter molecule. In this instance, the antisense molecule can be complementary to a portion of the entire mRNA molecule encoding the channel. These shorter antisense molecules are capable of hybridizing to the mRNA encoding the entire molecule, and preferably consist of about twenty to about one hundred nucleotides.
- antisense molecules can be used to reduce levels of pancreatic T- type calcium channel, by introducing into cells an RNA or smgle-stranded DNA molecule that is complementary to at least a portion of the mRNA of the channel (i.e. by introducing an antisense molecule) .
- the antisense molecule can base-pair with the mRNA of the channel, preventing translation of the mRNA into protein.
- an antisense molecule to the channel can prevent translation of mRNA encoding the channel into a functional channel protein.
- an antisense molecule complementary to at least a portion of mRNA encoding a pancreatic T-type calcium channel can be used to decrease expression of a functional channel.
- a cell with a first level of expression of a functional pancreatic T-type — calcium channel is selected, and then the antisense molecule is introduced into the cell.
- the antisense molecule blocks expression of functional pancreatic T- type calcium channel, resulting m a second level of expression of a functional pancreatic T-type calcium channel m the cell .
- the second level is less than the initial first level.
- Antisense molecules can be introduced into cells by any suitable means.
- the antisense RNA molecule is injected directly into the cellular cytoplasm, where the RNA interferes with translation.
- a vector may also be used for introduction of the antisense molecule into a cell.
- Such vectors include various plasmid and viral vectors.
- the invention further provides a special category of antisense RNA molecules, known as ribozymes, having recognition sequences complementary to specific regions of the mRNA encoding the pancreatic T-type calcium channel .
- Ribozymes not only complex with target sequences via complementary antisense sequences but also catalyze the hydrolysis, or cleavage, of the template mRNA molecule. Examples, which are not intended to be limiting, of suitable regions of the mRNA template to be targeted by ribozymes are any of the homologous regions identified by comparing the various T-type calcium channels, and particularly pancreatic ⁇ -cell T-type channels .
- Expression of a ribozyme m a cell can inhibit gene expression (such as the expression of a pancreatic T-type calcium channel) . More particularly, a ribozyme having a recognition sequence complementary to a region of a mRNA encoding a pancreatic T-type calcium channel can be used — to decrease expression of pancreatic T-type calcium channel . A cell with a first level of expression of pancreatic T-type calcium channel is selected, and then the ribozyme is introduced into the cell . The ribozyme m the cell decreases expression of pancreatic T-type calcium channel m the cell, because mRNA encoding the pancreatic T-type calcium channel is cleaved and cannot be translated.
- Ribozymes can be introduced into cells by any suitable means.
- the ribozyme is injected directly into the cellular cytoplasm, where the ribozyme cleaves the mRNA and thereby interferes with translation.
- a vector may be used for introduction of the ribozyme into a cell.
- Such vectors include various plasmid and viral vectors (note that the DNA encoding the ribozyme does not need to be "incorporated" into the genome of the host cell; it could be expressed m a host cell infected by a viral vector, with the vector expressing the ribozyme, for instance) .
- ribozymes and their use see Sarver et al . 1990, Chrisey et al . 1991, Rossi et al . 1992, and Ch ⁇ stoffersen et al . 1995.
- the nucleic acid molecules of the subject invention can be expressed m suitable host cells using conventional techniques. Any suitable host and/or vector system can be used to express the pancreatic T-type calcium channel. For m vitro expression, Xenopus oocytes are preferred. For m vivo expression, the most suitable host cell is a pancreatic ⁇ -cell. Techniques for introducing the nucleic acid molecules into the host cells may involve the use of expression vectors which comprise the nucleic acid molecules. These expression vectors (such as plasmids and viruses; viruses including bacteriophage) can then be- used to introduce the nucleic acid molecules into suitable host cells.
- expression vectors such as plasmids and viruses; viruses including bacteriophage
- DNA encoding the pancreatic T-type calcium channel can be injected into the nucleus of a host cell or transformed into the host cell using a suitable vector, or mRNA encoding the pancreatic T-type calcium channel can be injected directly into the host cell, m order to obtain expression of pancreatic T-type calcium channel m the host cell .
- Various methods are known m the art for introducing nucleic acid molecules into host cells. One method is micromj ection, m which DNA is injected directly into the nucleus of cells through fine glass needles (or RNA is injected directly into the cytoplasm of cells) .
- DNA can be incubated with an inert carbohydrate polymer (dextran) to which a positively charged chemical group (DEAE, for diethylammoethyl) has been coupled.
- DEAE positively charged chemical group
- the DNA sticks to the DEAE-dextran via its negatively charged phosphate groups.
- These large DNA- containing particles stick m turn to the surfaces of cells, which are thought to take them m by a process known as endocytosis. Some of the DNA evades destruction m the cytoplasm of the cell and escapes to the nucleus, where it can be transcribed into RNA like any other gene m the cell.
- cells efficiently take m DNA m the form of a precipitate with calcium phosphate.
- DNA enters through the holes directly into the cytoplasm, bypassing the endocytotic vesicles through which they pass m the DEAE-dextran and calcium phosphate procedures.
- DNA can also be incorporated into artificial lipid vesicles, liposomes, which fuse with the— cell membrane, delivering their contents directly into the cytoplasm.
- DNA is absorbed to the surface of tungsten micropro ectiles and fired into cells with a device resembling a shotgun.
- U.S. Patent No. 4,237,224 to Cohen and Boyer describes the production of expression systems m the form of recombinant plasmids using restriction enzyme cleavage and ligation with DNA ligase. These recombinant plasmids are then introduced by means of transformation and replicated m unicellular cultures including procaryotic organisms and eucaryotic cells grown m tissue culture. The DNA sequences are cloned into the plasmid vector using standard cloning procedures known m the art, as described by Sambrook et al . (1989) .
- Host cells into which the nucleic acid encoding the pancreatic T-type calcium channel has been introduced can be used to produce (i.e. to functionally express) the pancreatic T-type calcium channel.
- the function of the encoded pancreatic T-type calcium channel can be assayed — according to methods known m the art (Wang et al . 1996) .
- the invention further provides a method of screening a substance (for example, a compound or inhibitor) for the ability of the substance to modify T- type calcium channel function.
- the method comprises introducing a nucleic acid molecule encoding the pancreatic T-type calcium channel into a host cell, and expressing the pancreatic T-type calcium channel encoded by the molecule m the host cell.
- the cell is then exposed to a substance and evaluated to determine if the substance modifies the function of the T-type calcium channel. From this evaluation, substances effective m altering the function of the T-type calcium channel can be found.
- Such agents may be, for example, calcium channel inhibitors, agonists, or antagonists (for example, mibefradil and mibefradil analogues, amiloride, N ⁇ Cl 2 , antisense molecules, and second messengers) .
- the evaluation of the cell to determine if the substance modifies the function of the T-type calcium channel can be by any means known m the art .
- the evaluation can comprise the direct monitoring of expression of T-type calcium channel the host cell, or the evaluation can be indirect and comprise the monitoring of calcium transport by the channel (such as by the methods disclosed by Wang et al . 1996) .
- the nucleic acid molecules of the subject invention can be used either as probes or for the design of primers to obtain DNA encoding other pancreatic T-type calcium channels by either cloning and colony/plaque hybridization or amplification using the polymerase chain- reaction (PCR) .
- PCR polymerase chain- reaction
- Specific probes derived from SEQ ID NO : 1 can be employed to identify colonies or plaques containing cloned DNA encoding a member of the pancreatic T-type calcium channel family using known methods (see Sambrook et al . 1989) .
- One skilled m the art will recognize that by employing such probes under high stringency conditions (for example, hybridization at 42°C with 5X SSPC and 50% formamide, washing at 50-65°C with 0.5X SSPC), sequences having regions which are greater than 90% homologous or identical to the probe can be obtained.
- Sequences with lower percent homology or identity to the probe can be obtained by lowering the stringency of hybridization and washing (e.g., by reducing the hybridization and wash temperatures or reducing the amount of formamide employed) .
- the method comprises selection of a DNA molecule encoding a pancreatic T-type calcium channel, or a fragment thereof, the DNA molecule having a nucleotide sequence as shown m SEQ ID NO : 1 , and designing an oligonucleotide probe for pancreatic T-type calcium channel based on SEQ ID NO : 1.
- a genomic or cDNA library of an organism is then probed with the oligonucleotide probe, and clones are obtained from the library that are recognized by the oligonucleotide probe so as to obtain DNA encoding another pancreatic T-type calcium channel .
- Specific primers derived from SEQ ID NO : 1 can be used in PCR to amplify a DNA sequence encoding a member of the pancreatic T-type calcium channel family using known methods (see Innis et al . 1990) .
- One skilled in the art will recognize that by employing such primers — under high stringency conditions (for example, annealing at 50-60°C, depending on the length and specific nucleotide content of the primers employed) , sequences having regions greater than 75% homologous or identical to the primers will be amplified.
- the method comprises selection of a DNA molecule encoding pancreatic T-type calcium channel, or a fragment thereof, the DNA molecule having a nucleotide sequence as shown in SEQ ID NO : 1 , designing degenerate oligonucleotide primers based on regions of SEQ ID NO:l, and employing such primers in the polymerase chain reaction using as a template a DNA sample to be screened for the presence of pancreatic T-type calcium channel -encoding sequences.
- the resulting PCR products can be isolated and sequenced to identify DNA fragments that encode polypeptide sequences corresponding to the targeted region of pancreatic T-type calcium channel.
- the invention thus further provides an isolated nucleic acid molecule encoding a pancreatic T-type calcium channel, the nucleic acid molecule encoding a first amino acid sequence having at least 90% amino acid identity to a second amino acid sequence, the second amino acid sequence as shown in SEQ ID NO: 2.
- the first amino acid sequence has at least 95%, 96%, 97%, 98%, or 99% ammo acid identity to SEQ ID NO: 2.
- the invention further provides an isolated DNA oligomer capable of hybridizing to the nucleic acid molecule encoding pancreatic T-type calcium channel ⁇ according to the subject invention.
- Such oligomers can be used as probes m a method of detecting the presence of pancreatic T-type calcium channel m a sample. More particularly, a sample can be contacted with the DNA oligomer and the DNA oligomer will hybridize to any pancreatic T-type calcium channel present m the sample, forming a complex therewith. The complex can then be detected, thereby detecting presence of pancreatic T-type calcium channel the sample. The complex can be detected using methods known m the art.
- the DNA oligomer is labeled with a detectable marker so that detection of the marker after the DNA oligomer hybridizes to any pancreatic T-type calcium channel m the sample (wherein non-hybridized DNA oligomer has been washed away) is detection of the complex. Detection of the complex indicates the presence of pancreatic T-type calcium channel m the sample. As will be readily apparent to those skilled m the art, such a method could also be used quantitatively to assess the amount of pancreatic T-type calcium channel m a sample .
- the oligomers can be labeled with, for example, a radioactive isotope, biotin, an element opaque to X-rays, or a paramagnetic ion.
- Radioactive isotopes are commonly used and are well known to those skilled m the art. Representative examples include mdium-lll, technet ⁇ um-99m, and ⁇ odme-123.
- Biotm is a standard label which would allow detection of the biotm labeled oligomer with avidin.
- Paramagnetic ions are also commonly used and include, for example, chelated metal ions of chromium (III) , manganese (II) , and iron (III) .
- the labeled DNA oligomer can be imaged using methods known to those skilled m the art. Such imaging methods include, but are not limited to, X- — ray, CAT scan, PET scan, NMRI , and fluoroscopy.
- Other suitable labels include enzymatic labels (horseradish peroxidase, alkaline phosphatase, etc.) and fluorescent labels (such as FITC or rhodamine, etc.) .
- the invention further provides an isolated pancreatic T-type calcium channel protein.
- the protein is preferably encoded by a nucleotide sequence as shown m SEQ ID NO : 1.
- the protein preferably has an ammo acid sequence as shown SEQ ID NO : 2.
- pancreatic T-type calcium channel protein encoded by a first ammo acid sequence having at least 90% ammo acid identity to a second ammo acid sequence, the second ammo acid sequence as shown m SEQ ID NO : 2.
- the first ammo acid sequence has at least 95%, 96%, 97%, 98%, or 99% ammo acid identity to SEQ ID NO: 2.
- the pancreatic T-type calcium channel molecule of the subject invention can include a leader sequence for targeting of the pancreatic T-type calcium channel protein to the desired part of a cell.
- a met residue may need to be added to the ammo terminal of the ammo acid sequence of the mature pancreatic T-type calcium channel protein (i.e., added to SEQ ID NO: 2) or an ATG added to the 5' end of the nucleotide sequence (i.e., added to SEQ ID NO:l), m order to express the channel m a host cell.
- the met version of the mature channel is thus specifically intended to be covered by reference to SEQ ID NO : 1 or SEQ ID NO: 2.
- the invention further provides an antibody or fragment thereof specific for the pancreatic T-type calcium channel of the subject invention.
- Antibodies of — the subject invention include polyclonal antibodies and monoclonal antibodies capable of binding to the pancreatic T-type calcium channel, as well as fragments of these antibodies, and humanized forms. Humanized forms of the antibodies of the subject invention may be generated using one of the procedures known m the art such as chime ⁇ zation . Fragments of the antibodies of the present invention include, but are not limited to, the Fab, the F(ab') 2 , and the Fc fragments.
- the invention also provides hybridomas which are capable of producing the above-described antibodies.
- a hybridoma is an immortalized cell line which is capable of secreting a specific monoclonal antibody.
- the protein which is used as an immunogen may be modified or administered m an adjuvant m order to increase the protein's antigenicity.
- Methods of increasing the antigenicity of a protein include, but are not limited to, coupling the antigen with a heterologous protein (such as a globulin or beta-galactosidase) or through the inclusion — of an adjuvant during immunization.
- spleen cells from the immunized animals are removed, fused with myeloma cells, such as SP2/0-Ag 15 myeloma cells, and allowed to become monoclonal antibody producing hybridoma cells.
- myeloma cells such as SP2/0-Ag 15 myeloma cells
- any one of a number of methods well known m the art can be used to identify the hybridoma cell which produces an antibody with the desired characteristics. These include screening the hybridomas with an ELISA assay, western blot analysis, or radioimmunoassay (Lutz et al . 1988) .
- Hybridomas secreting the desired antibodies are cloned and the class and subclass are determined using procedures known the art (Campbell 1984) .
- antibody containing antisera is isolated from the immunized animal and is screened for the presence of antibodies with the desired specificity using one of the above-described procedures.
- the present invention further provides the above- described antibodies m detectably labeled form.
- Antibodies can be detectably labeled through the use of radioisotopes, affinity labels (such as biotm, avidin, etc.), enzymatic labels (such as horseradish peroxidase, alkaline phosphatase, etc.), fluorescent labels (such as FITC or rhodamine, etc.), paramagnetic atoms, etc.
- radioisotopes such as biotm, avidin, etc.
- enzymatic labels such as horseradish peroxidase, alkaline phosphatase, etc.
- fluorescent labels such as FITC or rhodamine, etc.
- paramagnetic atoms etc.
- the labeled antibodies or fragments thereof of the present invention can be used for m vitro, m vivo, and m situ assays to identify cells or tissues which express pancreatic T-type calcium channel, to identify samples containing pancreatic T-type calcium channel, or to — detect the presence of pancreatic T-type calcium channel m a sample. More particularly, the antibodies or fragments thereof can thus be used to detect the presence of pancreatic T-type calcium channel m a sample, by contacting the sample with the antibody or fragment thereof .
- the antibody or fragment thereof binds to any pancreatic T-type calcium channel present m the sample, forming a complex therewith.
- the complex can then be detected, thereby detecting the presence of pancreatic T- type calcium channel m the sample.
- a method could also be used quantitatively to assess the amount of pancreatic T-type calcium channel m a sample.
- such an antibody can also be used to decrease levels of functional T type calcium channels, by blocking the channel.
- Such antibodies can therefore be used m the methods of the subject invention to modify levels of functional T type calcium channels m pancreatic beta cells.
- a composition comprising the pancreatic T-type calcium channel protein and a compatible carrier.
- tissues or cells are contacted with or exposed to the composition of the subject invention or a compound.
- to "contact" tissues or cells with or to “expose” tissues or cells to a composition or compound means to add the composition or compound, usually m a liquid carrier, to a cell suspension or tissue sample, either m vitro or ex vivo, or to administer the composition or compound to cells or tissues withm an animal (including humans) .
- methods of modifying insulin secretion by pancreatic beta cells for therapeutics, methods of treating — type II diabetes, methods of modifying basal calcium levels m cells, methods of modifying the action potential of L type calcium channels m cells, methods of modifying pancreatic beta cell death, methods of modifying pancreatic beta cell proliferation, and methods of modifying calcium influx through L type calcium channels m cells, each of the methods comprising modifying levels of functional T type calcium channels m the cells, are provided.
- the formulation of therapeutic compositions and their subsequent administration is believed to be withm the skill m the art.
- compositions m accordance with the invention commonly m a pharmaceutically acceptable carrier, m amounts and for periods which will vary depending upon the nature of the particular disease, its severity and the patient's overall condition.
- the pharmaceutical compositions of the present invention may be administered m a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, mtranasal, transdermal), oral or parenteral .
- Parenteral administration includes intravenous drip or infusion, subcutaneous, mtraperitoneal or intramuscular injection, pulmonary administration, e.g., by inhalation or insufflation, or mtrathecal or mtravent ⁇ cular administration.
- Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
- Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or — desirable.
- Coated condoms, gloves and the like may also be useful .
- compositions for oral administration include powders or granules, suspensions or solutions m water or non- aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be desirable.
- compositions for parenteral, mtrathecal or mtravent ⁇ cular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.
- cationic lipids may be included the formulation to facilitate uptake.
- One such composition shown to facilitate uptake is LIPOFECTIN (BRL, Bethesda MD) .
- Dosing is dependent on severity and responsiveness of the condition to be treated, with course of treatment lasting from several days to several months or until a cure is effected or a diminution of disease state is achieved.
- Optimal dosing schedules can be calculated from measurements of drug accumulation m the body. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual compositions, and can generally be calculated based on IC 50 's or EC 50 ' s m m vitro and m vivo animal studies.
- a dose m mg/kg is routinely calculated.
- the methods of the subject invention are based on the discovery that regulation of T type calcium channels directly modifies basal calcium levels m cells, which in— turn regulates L type calcium channel activity, which m turn regulates insulin secretion and cell death, which m turn treats type II diabetes.
- the methods of the subject invention are further based on the discovery that regulation of T type calcium channels directly affects basal and glucose-induced insulin secretion.
- T type calcium channels belong to the family of low voltage activated calcium channels.
- Modifying (increasing or decreasing) "levels" of functional T type calcium channels refers to modifying expression of the T type calcium channel gene, modifying activity of the T type calcium channel such as by inhibiting the function of the channel, and/or modifying the formation of active membrane-spanning T type calcium channels.
- “functional” refers to the synthesis and any necessary post-translational processing of a calcium channel molecule m a cell so that the channel is inserted properly m the cell membrane and is capable of conducting calcium ions m accordance with a low voltage activated channel.
- the invention thus provides a method of modifying msulm secretion by pancreatic beta cells, the method comprising modifying levels of T type calcium channels m the pancreatic beta cells.
- Levels of T type calcium channels m the pancreatic beta cells can be modified by various methods, at the gene and protein and "functional calcium channel" levels.
- the levels are modified by modifying T type calcium channel gene expression of the T type calcium channel m the cells. This can be accomplished by exposing the cells to a compound which modifies T type calcium channel gene expression of the calcium channel .
- the compound could be, for example, an antisense oligonucleotide targeted to the T type calcium channel — gene.
- the compound which modifies T type calcium channel gene expression of the T type calcium channel could be a ribozyme.
- T type calcium channel gene expression could also involve site-directed mutagenesis of the T type calcium channel gene to prevent expression of the T type calcium channel, or various gene therapy techniques .
- Levels, m particular activity, of T type calcium channels m the cell can also be modified by exposing the cells to an inhibitor of the T type calcium channel .
- inhibitors include, for example, mibefradil, mibefradil analogs, amiloride, N ⁇ Cl 2 , and second messengers which regulate activity of the T type calcium channels.
- Other inhibitors of the T type calcium channel could also readily be identified by screening methods (including the method described above) .
- peptide inhibitors could also be identified with screening methods (for example, using phage display libraries and other peptide screening methods) .
- alkyl is meant to include linear alkyls, particularly C1-C12 linear alkyls (e.g., methyl, ethyl, n-propyl, n-pentyl, n-hexyl, n-heptyl, n-octyl, n-nonyl, and the like), branched alkyls, particularly C1-C12 branched alkyls (e.g., isobutyl, isopentyl, neopentyl , hex-2-yl, hex-3-yl, hept-2-yl, hept-3-yl, and the like), and cycloalkyls, particularly C1-C8 cycloalkyls (e.g., cyclopentyl, cyclohe
- alkyl groups can be substituted or unsubstituted.
- suitable substituents include, for example, aryl groups, halogen atoms, hydroxy groups, alkoxy groups, carboxylic acid groups, amine groups, and the like, as well as combinations of these substituents.
- Mibefradil analogs which are particularly well suited to blocking (inhibiting) the activity of T- type calcium channels but not blocking the activity of L- type calcium channels are those having the formula:
- R is an unsubstituted alkyl group or a substituted alkyl group which does not contain an alkoxy substituent.
- "Mibefradil analogs” are also meant to include compounds having the above formulae which are substituted at other positions m the structure, for example, on the benzimidazole phenyl moiety, at a benzimidazole nitrogen, at other positions of the tetrahydronaphthyl ring, etc. Also included withm the meaning of "mibefradil analogs" are compounds having the above formulae m which the F is replaced with another substituent, such as another halogen.
- misradial analogs are compounds having the above formulae m which the amme methyl group or the isopropyl group or both are replaced with other substituents, such as other alkyl moieties. Additionally, “mibefradil analogs” are meant to include those compounds which are gene ⁇ cally described and/or specifically disclosed m U.S. Patent No. 4,808,605, which is hereby incorporated by reference. Further, “mibefradil analogs” are meant to include pharmaceutically acceptable salts of the derivatives described above.
- Illustrative pharmaceutically acceptable salts are salts formed with hydrochloric acid, hydrobromic acid, nitric acid, sulphuric acid, phosphoric acid, citric acid, formic acid, maleic acid, acetic acid, succinic acid, tartaric acid, methanesulphonic acid, p-toluenesulphonic, and the like.
- Mibefradil analogs can be made by following the general procedures described m, for example, U.S. Patent Nos. 4,808,605, 5,910,606, 5,892,055, 5,811,557, —
- Levels of T type calcium channels m the cell can also be modified by exposing the cells to a compound which interferes with membrane T type calcium channel formation.
- Levels of functional T type calcium channel could also be modified by use of molecules which bind to transcription regulators of the T type calcium channel gene (such as the promoter region of the gene) .
- the invention further provides a method of treating type II diabetes a subject (human or animal) , the method comprising administering to the subject an amount of a compound effective to modify levels of T type calcium channels m the pancreatic beta cells of the subject.
- the compound may modify levels of T type calcium channels by modifying T type calcium channel gene expression of the calcium channel, or by inhibiting the T type calcium channel, or by interfering with membrane T type calcium channel formation.
- modulation means either inhibition or stimulation.
- This modulation can be measured m ways which are routine m the art, for example by Northern blot assay of mRNA expression, Western blot assay of protein expression, or calcium channel activity assay.
- the compounds and/or inhibitors used m the methods of the subject invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound/inhibitor which, upon administration to an animal including a human, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof.
- the disclosure is also drawn to prodrugs and ⁇ pharmaceutically acceptable salts of the compounds and/or inhibitors used m the subject invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents .
- the compounds and/or inhibitors for use m the invention may additionally or alternatively be prepared to be delivered m a prodrug form.
- prodrug indicates a therapeutic agent that is prepared m an inactive form that is converted to an active form (i.e., drug) withm the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions.
- pharmaceutically acceptable salts refers to physiologically and pharmaceutically acceptable salts of the compounds and/or inhibitors used m the subject invention: i.e., salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto.
- Drugs such as peptide drugs, which inhibit the T type calcium channel or which interfere with functional T type calcium channel formation can be identified by other methods also.
- a monoclonal antibody can be prepared which specifically hybridizes to the T type calcium channel, thereby interfering with activity and/or channel formation.
- the monoclonal (which is itself a compound or inhibitor which can be used m the subject invention) can be used to identify peptides capable of mimicking the inhibitory activity of the monoclonal antibody.
- One such method utilizes the development of epitope libraries and biopannmg of bacteriophage libraries. Briefly, attempts to define the binding sites for various monoclonal ⁇ antibodies have led to the development of epitope libraries. Parmley and Smith developed a bacteriophage expression vector that could display foreign epitopes on its surface (Parmley, S.F. & Smith, G.P., Gene 73:305-318 (1988)) .
- This vector could be used to construct large collections of bacteriophage which could include virtually all possible sequences of a short (e.g. six- ammo-acid) peptide. They also developed biopannmg, which is a method for affmity-purifymg phage displaying foreign epitopes using a specific antibody (see Parmley, S.F. & Smith, G.P., Gene 73:305-318 (1988); Cwirla, S.E., et al . , Proc Natl Acad Sci USA 87:6378-6382 (1990); Scott, J.K. & Smith, G.P., Science 249:386-390 (1990); Christian, R.B., et al . , J Mol Biol 227:711-718 (1992); Smith, G.P. & Scott, J.K., Methods m Enzymology 217:228- 257 (1993) ) .
- mimotopes peptide sequences which mimicked the epitope, i.e., sequences which did not identify a continuous linear native sequence or necessarily occur at all withm a natural protein sequence. These mimicking peptides are called mimotopes . In this manner, mimotopes of various binding— sites/protems have been found.
- sequences of these mimotopes do not identify a continuous linear native sequence or necessarily occur m any way m a naturally-occurring molecule, i.e. a naturally occurring protein.
- sequences of the mimotopes merely form a peptide which functionally mimics a binding site on a naturally- occurring protein.
- mimotopes are short peptides.
- the availability of short peptides which can be readily synthesized large amounts and which can mimic naturally-occurring sequences (i.e. binding sites) offers great potential application.
- mimotopes to a monoclonal antibody that recognizes T type calcium channels can be identified.
- the sequences of these mimotopes represent short peptides which can then be used m various ways, for example as peptide drugs that bind to T type calcium channels and decrease the activity of T type calcium channels.
- the peptide drugs can be chemically synthesized.
- INS-1 cells were cultured m RPMI 1640 medium containing 10% FBS, 25 U/ml penicillin, 25 mg/ml streptomycin and 50 ⁇ M mercaptoethanol m an atmosphere of 5% C0 2 m air, at 37°C for 2-5 days before recording .
- Islet cell preparation - Pancreases of Sprague-Dawley rats (Charles River Laboratory, Wilmington, MA) were removed after mtrapancreatic perfusion with 2 ml of Hanks' solution (Gibco BRL, Grand Island, NY) containing collagenase (4 mg/ml, Boehrmger — Mannheim, Indianapolis, IN), DNase I (10 ⁇ g/ml, Sigma, St.
- pancreatic tissue was incubated at 37 °C for 20 mm and then washed five times with enzyme-free Hanks' solution. Islets were picked up and treated with 0.1% pancreatm (Sigma) for five minutes at 37°C. Single cells were obtained by triturating the islets with plastic pipette tips and then they were transferred into 35 mm culture dishes. Cells were cultured m RPMI 1640 medium (Gibco BRL) containing 5 mM glucose, 10% FBS and P/S at 37°C, 5% C0 2 for 2-5 days before experiments.
- RNA - Total RNA was isolated from cultured INS-1 cells and from various freshly excised rat tissues by the guanidmium isothiocyanate/phenol procedure (Chomczynsk and Sacchi 1987) .
- Poly-A RNA was isolated from total RNA by two successive passes over an oligo (dT) -cellulose spin column (Ambion, Austin, TX) .
- Cloning of cDNA Encoding ⁇ l Subunit of T-type Ca 2+ channel m INS-1 - First strand cDNA was prepared using 2 ⁇ g of INS-1 cell mRNA and M-MLV reverse transc ⁇ ptase (Gibco BRL) with the poly-dT primers.
- the first 433 bp DNA fragment of the channel was deduced with PCR using the degenerate primers (forward) (SEQ ID NO: 6) 5'- TNGC(A/C/T)ATGGAG(C/A)GNCC(C/T) -3 ' and (backward) (SEQ ID NO:7) 5 ' -CTT(C/G/T)CCCTTGAA(G/C)A(G/A)CTG) -3 ' based on conserved voltage-dependent Ca 2+ channel a 1 subunit sequences m domain III .
- the 3'- and 5'- rapid amplifications of cDNA end-PCR were performed to obtain the entire gene of the ⁇ x subunit of the channel.
- the forward primer was an adapter primer
- the backward primer was (SEQ ID NO: 8) 5 ' -CCGCTGTCGGAGACCATGGAGACC-3 '
- the — forward primer was (SEQ ID NO: 9) 5'-
- the RT-PCR products were subcloned into pT-Adv Vector (Clontech) and dideoxynucleotide sequencing assay was performed with a dsDNA Cycle Sequencing System (Gibco BRL) .
- Tissue distribution The gene expression of T-type Ca 2+ channels deduced from ⁇ -cells was examined in rat brain, heart, kidney, and liver using an RT-PCR assay.
- the primers used for the RT-PCR were (SEQ ID NO: 10)
- the first PCR reaction was carried out in 5 tubes, each having a total volume of 50 ⁇ l: 5 ⁇ l 10X PCR reaction buffer, 1 ⁇ l dNTP (10 mM each), 2.2 ⁇ l Mg(0Ac) 2 (25 mM) , 1 ⁇ l API (10 ⁇ M) , 1 ⁇ l GSP1, 1 ⁇ l Advantage Genomic Polymerase Mix (5OX) , and 37.8 ⁇ l water.
- the following two-step cycle parameters were used: (Step 1) 7 cycles of denaturing at 94 °C for 25 sec, annealing and extension at 72°C for 4 min. (Step 2) 32 cycles of denaturing at 94°C for 25 sec., annealing and extension at 67°C for 4 min.
- the second PCR reaction was carried out under the reaction condition similar to the first PCR reaction except using AP2 , GSP2.
- the templates used were 1 ⁇ l of 1:50 dilution of each primary PCR reaction.
- the two step cycles were similar to the first PCR reaction except 5 cycles at the first step and 22 cycles at the second step. —
- Oocyte electrophysiology - cRNA transcripts were synthesized from BssH II linearized pT-Adv cDNA templates using T7 RNA polymerase (Ambion) . Defolliculated Xenopus laevis were injected with 25 ng pT-Adv cRNA. Three to five days after injection, two-electrode voltage-clamp recording was performed using a Warner OC-725C amplifier (Warner Instrument Corp., Hamden, CT) . Data were acquired and analyzed with Pulse/PulseFit software (HEKA, Lambrecht/Pfalz, Germany) .
- the bath solution contained the following: 40 mM Ca(OH) 2 , 50 mM NaOH, 2 mM TEA-C1, ImM KOH, 0.1 mM EDTA and 5 mM HEPES, adjusted to pH 7.4 with methanesulphonate .
- Boltzmann fits were calculated using Prism (GraphPad) . Results are presented as mean ⁇ s.d. unless otherwise stated.
- ⁇ -cell Electrophysiological recording The whole-cell recordings were carried out by the standard "giga-seal" patch clamp technique (Hamill et al . ) .
- the whole-cell recording pipettes were made of hemocapillaries (Warner) , pulled by a two-stage puller (PC-10, Narishige International, New York, NY), and heat polished with a microforge (MF200-1, World Precision Instruments, Sarasota, FL) before use. Pipette resistance was in the range of 2-5 M ⁇ in the internal solution.
- the recordings were performed at room temperature (22-25°C) .
- Currents were recorded using an EPC-9 patch-clamp amplifier (HEKA) and filtered at 2.9 kHz. Data were acquired with Pulse/PulseFit software (HEKA) . Voltage-dependent currents were corrected for linear leak and residual capacitance by using an on-line P/n subtraction paradigm.
- Drugs - Mibefradil (IS, 2S) -2- [2- [ [3 - (2 - Benzimidazolyl) propyl] methyl -amino] ethyl] -6-fluoro- 1 , 2 , 3 , 4-tetrahydro-l-isopropyl-2-napthyl methoxy-acetate — dihydrochloride) was kindly provided by Dr. J.-P. Clozel (Hoffmann LaRoche, Basel, Switzerland), and can be synthesized according to the methods disclosed in U.S. Patent Nos. 5,892,055, 5,811,557, 5,811,556, and 5,808,088.
- U.S. Patent No. 4,808,605 describes mibefradil compounds suitable for use in the subject invention.
- the intracellular solution contained (in mM) : 130 N-methyl-D- glucamine, 20 EGTA (free acid), 5 bis (2 -aminophenoxy) ethane-N, N, N', N' -tetraacetate (BAPTA) , 10 HEPES, 6 MgCl 2 , 4 Ca(OH) 2 , pH was adjusted to 7.4 with methanesulfonate. 2 mM Mg-ATP was included m the pipette solution to minimize rundown of L-type Ca 2+ currents.
- the extracellular solution contained (m mM) : 26 Sucrose, 30 TEA-C1, 10 HEPES, 5 KCl, 2 CaCl 2 , MgCl 2 , pH 7.3.
- the pipette solution contained (m mM) : 65 CsOH, 65 CsMS , 20 sucrose, 10 HEPES, 10 MgCl 2 , 1 Ca(OH) 2 , pH 7.4.
- Mass Spectrometric Analysis A VG 70-250 SEQ instrument (VG Analytical, Manchaster, UK) was used with fast atom bombardment (FAB) lonization mode to obtain mass spectra of the mibefradil and dm-mibefradii .
- INS-1 cells were treated with 20 ⁇ M mibefradil for various lengths of time under each experimental condition.
- the cell pellets were collected after washing three times with PBS and resuspended m 0.5 ml media for mass spectrometric analysis.
- 20 ⁇ l internal standard solution 40 ⁇ M verapamil, MW:454
- 5 ⁇ l glycerol was added, and 4 ⁇ l of this mixture was used for FAB-MS.
- Several positive ion spectra were recorded m the mass range m/z 750-100 at a mass resolution of 1000, and a scan speed of 2 second/decade.
- m/z 496 was the dominant ion (M+H) + accompanied with a less intense sodiated molecular ion m/z 518.
- concentrations of the mibefradil and hydrolyzed mibefradil were obtained by comparing the intensities of m/z 496 and 424 were to the intensity of m/z 455.
- a standard solution of 50 ⁇ M drug was subjected to mass spectrometric analysis.
- cytosolic and membrane components After washing out mibefradil from the bath solution, the cells were collected and the membranes were broken down by vortexmg the cells m a solution containing 5% acetic ac ⁇ d/CH 3 CN. The mixture was then spun and the supernatant collected and defined as non-membrane associated components. Pellets were re-suspended m 5 x volume of NaOH (10 N) : methanol (1:7) solution at 37°C for 5 mm. The mixture was neutralized with 0.5 M HCI and spun down.— The remaining pellet and the supernatant were collected separately.
- the subject invention provides a cDNA encoding a T-type Ca 2+ channel ot 1 subunit derived from the rat insulin secreting cell line, INS-1, which has been identified and sequenced.
- the sequence of the cDNA indicates a protein composed of 2288 ammo acids (SEQ ID NO:2) , sharing 96.3% identity to the neuronal T-type Ca 2+ channel cx 1 subunit (o ⁇ G) .
- the transmembrane domains of the protein are highly conserved but the isoform contains three distinct regions as well as 10 single ammo acid substitutions m other regions. Sequencing rat genomic DNA revealed that this is an alternative splice isoform of C-jG.
- the T-type Ca 2+ channel gene deduced from ⁇ -cells shares 96.3% amino acid identity with x G, the neuronal isoform of T-type Ca 2+ channel (Perez-Reyes et al . 1998) .
- the four intramolecular homologous transmembrane domains of ⁇ -cell T-type Ca 2+ channel ⁇ x subunit are identical (except glycine 1667) to a x G, with each repeat containing six putative membrane-spanning regions (S1-S6) and a pore-forming region (P-loop) .
- the other highly conserved region is located at the intracellular loop between repeat I and II, where a section of histidine-rich chain is present in the ⁇ -cell derived T-type Ca 2+ channel gene as well as in neuronal and cardiac T-type Ca 2+ channel genes.
- This structure in the loop ⁇ n has not been observed in the protein sequences of known high voltage activated Ca 2+ channels.
- the T-type Ca 2+ channel gene derived from ⁇ -cells contains three unique regions that differ from the amino acid sequence of x G.
- These regions are located at the N-terminal amino acids (aal-34 of SEQ ID NO:2), — intracellular loop I II-Iir (aa971-994 of SEQ ID NO : 2 ) and intracellular loop h I ⁇ _ IV (aal570-1588 of SEQ ID NO : 2 ) .
- nucleotide sequences at this region are almost identical except for 4 single nucleotide insertions which are shown in Fig. IA. These four single nucleotide insertions determine a different start codon as well as those of the amino acid sequences.
- T-type Ca 2+ channel isoform deduced from INS-1 and ⁇ G a section of Sprague-Dawley rat genomic DNA sequence containing the introns and exons between 4845 and 5256 was identified. As shown in Fig. IB, an exon was found that encodes the ⁇ x G fragment SKEKQMA (SEQ ID NO: 5) as well as an exon that encodes fragment 4869-4922 of the INS-1 variant. This region also contains 8.5 kilobases (kb) of intron sequence.
- the T-type Ca 2+ channel ⁇ x subunit cloned from INS-1 and ⁇ x G are alternative splice isoforms of the same gene .
- the genomic DNA sequence was also used to examine the two nucleotide discrepancy between the ⁇ x G cDNA and the isoform cloned from INS-1.
- the data show that the genomic nucleotide sequence encoding amino acid 1667 is GGC (glycine) , which is the same as the cDNA of a 1 subunit cloned from INS-1 and the corresponding residue in ⁇ H, but is different from ⁇ x G (GCG, alanine) .
- GGC glycine
- Six correspond to the amino acids found in the analogous position of ⁇ H: cysteine 1088, glycine 1667, alanine 1700, aspartic acid 1735, threonine 1812, and leucine 1813.
- T-type Ca 2+ channels deduced from ⁇ -cells and from neurons
- expression of the ⁇ -cell T-type Ca 2+ channel was found in rat brain, heart and kidney, but was absent from liver. Both ⁇ -G and the splice form were detected in rat islets and INS-1 cell preparations using RT-PCR. No ⁇ x H was detected.
- Fig. 2B The voltage-dependent activation (Fig. 2C) and steady-state inactivation (Fig. 2D) were fitted with Boltzmann equation. The calculated V 1/2 ' s were -23.8 mV and -45.6 mV for activation and inactivation, respectively; and k ' s were 5.3 and -6.0 for activation and inactivation, respectively.
- SEQ ID NO:l The nucleotide cDNA (SEQ ID NO:l) and amino acid (SEQ ID NO : 2 ) sequences of rat pancreatic T-type calcium channel were determined.
- SEQ ID NO : 3 is the nucleotide sequence beyond the coding region, while SEQ ID NO : 4 includes SEQ ID NO: 2.
- Glucose stimulated insulin release is Ca 2+ dependent process, involving closure of the ATP-sensitive potassium-" channels, depolarization and opening of the voltage- dependent Ca 2+ channels.
- ⁇ -cells are electrically silent with a resting membrane potential of about -70 mV.
- Raising external glucose produces a slow depolarization, the extend dependent upon the glucose concentration.
- depolarization is sufficient to reach the threshold potential (-50 mV) at which electrical activity is initiated.
- m Fig. 12 A simple model for glucose-stimulated insulin secretion is summarized m Fig. 12.
- the resting membrane potential of ⁇ -cells is principally determined by the activity of the K-ATP channel.
- the intracellular ATP or ATP:ADP ratio
- the intracellular ATP increases which leads to the closure of K- ATP channels and membrane depolarization.
- the increased calcium influx leads to a rise m [Ca 2+ ] 1 and consequently insulin secretion.
- Rat and human pancreatic ⁇ -cells are equipped with L-type and T-type Ca 2+ channels.
- the physiological function of T-type Ca + channels m ⁇ -cells msulm- secretion has been demonstrated. These channels facilitate exocytosis by enhancing electrical activity m these cells.
- L-type and T-type Ca 2+ channels under normal conditions, work m concert promoting the rise m [Ca 2+ ] , during glucose-stimulated insulin secretion.
- T-type Ca 2+ channels are, at least m part, responsible for the hyper-responsiveness of insulin secretion to non-glucose depolarizing stimuli m GK rat, and m rat with NIDDM induced by neonatal — injection of streptozotocm.
- T-type calcium channels over time will ultimately lead to an elevation of basal Ca 2+ through its window current properties. Therefore, there is a dual effect of T-type Ca 2+ channels m ⁇ -cells depending upon channel number and membrane potential.
- Pharmacologically antagonizing T-type calcium channels is an appropriate treatment protocol for alleviating both insulin resistance and enhancement of insulin secretion m NIDDM patients.
- NIDDM pathogenesis is complex and the disease progression occurs m phases.
- An enhanced ⁇ -cell responsiveness provokes and initiates the disease process. It is unclear as to what the actual enhanced activity is and what the triggering mechanisms are for this first phase. It may be an increased secretory response or an increase m ⁇ -cell mass.
- hypermsulmemia there is clearly an enhancement of ⁇ -cell activity detected by both basal and postprandial elevated insulin levels denoted as hypermsulmemia . Consequently, a resulting msulm resistance occurs, phase II, particularly m insulin responsive tissues (muscle, liver, kidney, fat) that function to reduce glucose levels m the blood.
- a decrease m msulm sensitivity will account for an increase blood glucose, causing the ⁇ -cells to secrete even more msulm to compensate and because of this vicious cycle, full blown NIDDM, marked by an inevitable defect m msulm release, hyperglycemia and msulm resistance, will characterize the final stage of the disease process.
- Each phase of the disease may be characterized by an alteration m [Ca 2+ ] x , and each phase can be treated by a T-type calcium channel antagonist.
- the electrical ⁇ -cell— is equipped with two types of voltage-dependent calcium channels, L-type and T-type calcium channels.
- L-type calcium channels activated at high voltages, having large unitary conductance, and dihydropy ⁇ dme- sensitive, are considered the major pipeline for calcium influx into the ⁇ -cell (especially at high voltage depolarization) .
- T-type calcium channels, activated at low voltages, with small unitary conductance, and dihydropyridme- msensitive, are important for maintaining basal [Ca 2+ ] , (Fig.
- T-type calcium channels normally facilitate msulm secretion m ⁇ -cells by enhancing cell electrical activity. This modulatory function of T-type calcium channels m msulm secretion is significant during phase I prior to onset of diabetes. Antagonizing these T-type calcium channels will decrease ⁇ -cell hyper- responsiveness and consequent hypermsulmemia arresting the pathogenic pathways that lead to NIDDM.
- a T-type calcium channel blocker is still the appropriate treatment protocol.
- the msulm responsive tissues those that are primarily responsible for taking up glucose for re-establishing euglycemia, have elevated basal [Ca 2+ ] . during hypermsulmemic conditions. Indeed, it is the elevated basal [Ca 2+ ] . that precipitates the decrease m msulm sensitivity of these tissues and it is now known that most of these msulm responsive tissues express T-type calcium channels.
- a T-type calcium channel blocker will reduce the basal [Ca 2+ ] x and alleviate the decreased msulm sensitivity.
- NIDDM Once NIDDM has manifested, it is characterized by altered glucose metabolism, a result of abnormal glucose stimulus-secretion responsiveness of ⁇ -cells.
- ⁇ -cell desensitization to glucose is the principal secretory defect of NIDDM.
- L-type and T-type calcium channels under normal conditions, work m concert promoting the rise m [Ca 2+ ] , during glucose-stimulated msulm secretion. In NIDDM, this partnership is broken and the necessary rise m [Ca 2+ ] . for msulm secretion is compromised.
- the data herein indicates that L-type calcium channels are finely regulated by basal calcium levels (Figs. 9A-9D) .
- a very small rise m basal calcium will substantially decrease the L-type calcium current and severely reduce the depola ⁇ zation-mduced rise m [Ca 2+ ] , (Figs. 10 and 11) .
- the data herein also suggests that T- type calcium channels are a primary regulator of resting basal [Ca 2+ ] . m ⁇ -cells.
- the negative feedback regulation of T-type calcium channels by elevated [Ca 2+ ] . is absent (Figs. 9A-9D) .
- mibefradil has a potent inhibitory effect on T-type Ca 2+ current vascular smooth muscle cells.
- the data herein demonstrates that, m convention whole cell patch clamp configuration, mibefradil also blocks T-type Ca 2+ current m pancreatic ⁇ -cells.
- Mibefradil (1 ⁇ M) had been administered m the recording chamber at time zero (Fig. 13) , the control (no drug) showed "run down” .
- T-type Ca 2+ current is more sensitive to mibefradil than the L- type Ca 2+ current m pancreatic ⁇ -cells.
- the blockade of T-type Ca 2+ channels m ⁇ -cells with mibefradil is reversible.
- Fig. 14 demonstrates the reversibility of blockade of T-type Ca 2+ currents by mibefradil.
- a very little volume of mibefradil or N ⁇ Cl 2 was delivered near the recording cell.
- the drug then diffused away from the cell.
- the final concentration m the chamber was 1 nM .
- This experiment shows the inhibitory effect of mibefradil on T-type Ca 2+ current m pancreatic ⁇ -cells results from reversible interaction between the drug and the channel protein.
- T-type Ca 2+ channels could mediate a small, but sustained, Ca 2+ influx by means of their unique "window" current at voltages near resting membrane potentials.
- T- type Ca 2+ channels are opened and closed depending upon — the potentials across the cell membranes. This voltage dependency is illustrated in Fig. 15.
- the activation and inactivation curves represent the percentage of the channels in either open or closed states over a range of voltages. Unlike most of the voltage-dependent Na + channels or L-type Ca 2+ channels, the activation and inactivation curves of T-type Ca 2+ channels overlap at the certain range of low voltages (i.e. window) .
- window current provides a negative feedback regulation of [Ca 2+ ] . in ⁇ -cells.
- cells When cells are under an unhealthy condition, they may be slightly depolarized to activate window current, which elevates the basal [Ca 2+ ] . to protect the cells from further Ca 2+ influx through the L-type Ca 2+ channels. This process is reversible if the membrane potential is reset to the normal resting potential (-70 mV) .
- Mibefradil regulates basal [Ca 2+ ] , in pancreatic ⁇ -cells The data herein demonstrates the roles of T-type calcium currents in modulating basal [Ca 2+ ] , in INS-1 cells (Fig. 8) .
- [Ca 2+ ] 1 was directly measured by the ratio of fluorescence excitations at Ca 2+ -bound (380 nm) to unbound (340 nm) , and then the ratio was converted to the calcium concentration.
- the bath solution contained 10 mM NaCl, 4 mM KCl, 2 mM CaCl 2 , and 2 mM MgCl 2 .
- Mibefradil regulates basal msulm secretion
- NIT-1 The activation of T-type Ca 2+ channel at low voltage near the resting membrane potential of pancreatic ⁇ -cells suggests that the channels are responsible for the Ca 2+ influx required for msulm secretion under non-stimulus conditions.
- the NIT-1 cell line was chosen to demonstrate the effect of mibefradil on the basal msulm secretion.
- NIT-1 is a cell line derived from the ⁇ -cell of non-obese-diabetic mouse. This cell line expressed high levels of T-type Ca 2+ current.
- the data herein shows that 5 ⁇ M mibefradil reduced the basal msulm secretion to less than 40% of control (Fig. 17), indicating this drug is able to lower the high basal msulm secretion level seen during the earlier stage of NIDDM.
- NIT-1 cells were cultured m medium containing 3.3 mM glucose and preloaded with 2.5 ⁇ M Fluo- 3 AM. The numbers of spontaneous calcium elevated cells were counted and compared to the total cells being used for a 10 minute observation period. 10 ⁇ M NiCl 2 inhibited 90% of spontaneous elevation of basal Ca 2+ .
- NiCl 2 (30 ⁇ M) reduced the frequency of spontaneous calcium spikes immediately. This result suggests that either the T-type Ca 2+ channels alone or together with the L-type Ca 2+ channels are responsible for the transient spontaneous elevation of [Ca + ] 17 under conditions where no glucose is present . These spontaneous calcium spikes may contribute to basal insulin secretion and control of basal [Ca 2+ ] 1 .
- T type Ca 2+ may play two pathological roles in NIDDM.
- the NIDDM patients exhibit hyperinsulinemia and ⁇ -cell hyperexcitability . This may, at least in part, be due to increased activity of T type Ca + channel in ⁇ -cells.
- over-expressed T type Ca 2+ channel and membrane depolarization resulted from reduced generation of ATP, ⁇ and may set up a window current in ⁇ -cells that causes chronic elevation of basal Ca 2+ in the ⁇ -cells.
- the elevated basal Ca 2+ will reduce the L-type Ca 2+ activity and glucose induced insulin secretion. It has been shown that mibefradil prevented and reversed development of hyperinsulinemia in rat .
- T type Ca 2+ facilitated insulin secretion by enhancing the general excitability of pancreatic ⁇ -cells.
- activation of T type Ca 2+ channels will increase the firing frequency of the depolarizing spikes mediated by opening L type Ca 2+ channels (Fig. 19A) .
- Activation of T type Ca 2+ channel will also decrease the time of developing action potential elicited by up-threshold depolarizations (Fig. 19B) .
- NiCl 2 was administered to effectively block T type Ca 2+ channels.
- NiCl 2 caused a delay in the onset of an action potential and a decrease in number of action potentials.
- T type Ca 2+ current in glucose-induced insulin secretion
- INS-1 cells were incubated with 11.1 mM glucose and variable concentrations of NiCl 2 , and insulin release was measured.
- NiCl 2 reduced insulin secretion in a dose-dependent manner (Fig. 20A) .
- clonal insulin secreting cells HIT-T15, which did not consistently exhibit T type Ca 2+ current
- Fig. 20B clonal insulin secreting cells
- T type Ca 2+ channels facilitate insulin secretion by enhancing general excitability of ⁇ -cells
- the function of T type Ca 2+ channels is a doubled-edged sword.
- the function of the window current will become dominant and result in an elevation of basal Ca 2+ .
- High [Ca 2+ ] x may cause impairment of insulin release by inactivating L type Ca 2+ channels.
- L-type Ca 2+ channels are partially inactivated by [Ca 2+ ] 1 in non-stimulus condition in ⁇ -cells :
- the L type Ca 2+ current "runs-up", as the magnitude of the peak current increases over time in INS-1 cells (Fig. 21) .
- This phenomenon is a universal feature in these cells under the recording conditions used.
- the pipette solutions contained no ATP but did contain high concentrations of the calcium chelating agents BAPTA and EGTA. When the pipette solution contained high Ca 2+ , this run-up does not occur. Instead, a rapid run down occurs. The "run-up" phenomenon is likely due to calcium chelation inside the cells. T type Ca 2+ currents do not show this effect.
- Intracellular perfusion patch clamp experiments demonstrated that basal [Ca 2+ ] . regulates L type Ca 2* current amplitude in INS-1 cells:
- Intracellular perfusion of a solution containing high Ca 2+ causes a substantial reduction in the — L type Ca 2+ current.
- L type Ca 2+ currents were elicited by a voltage step to +10 mV from a holding potential of -80 mV.
- the [Ca 2+ ] was measured directly using fura-2 ratiometric fluorescence.
- the effect of a high [Ca 2+ ] x (272 nM) on the IV relationship is shown in Fig. 9B.
- the empirical K d obtained for calcium binding to fura-2 in the system was 296 + 20 nM.
- basal [Ca 2+ ] remains low, subsequent voltage stimulation with 50 KCl induces rapid and large calcium influx into the cell and these calcium changes are stereotyped upon repetitive stimulation when basal calcium is restored (Fig. 10) .
- the cell was repolarized by perfusion of the original 5 mM KCl solution. After repolarization, basal [Ca 2+ ] ⁇ slowly reset and then a second 50 KCl depolarization induced a similar [Ca 2+ ] . transient.
- basal [Ca 2+ ] ⁇ in INS-1 cells was artificially enhanced by pretreating the cells with the toxicant, streptozotocm. Though it is know that streptozotocm induces DNA strand breaks, it has also been shown to induce Ca 2+ channel activity m ⁇ - cells. The data shows that pretreatmg cells with 5 mM streptozotocm for 1 hour, followed by 3 hour recovery, — causes a two-fold increase m basal calcium (Fig. 22) . These cells when stimulated by 50 KCl had reduced calcium influx compared to control cells.
- mibefradil (Ro 40-5967) exerts a selective inhibitory effect on T-type Ca 2+ currents, although at higher concentrations it can antagonize high voltage-activated Ca 2+ currents.
- the action of mibefradil on Ca 2+ channels is use- and steady state-dependent and the binding site of mibefradil on L-type Ca 2+ channels is different from that of dihydropyridmes.
- mibefradil is shown to have an inhibitory effect on both T- and L-type Ca 2+ currents m msulm-secretmg cells.
- T-type Ca 2+ current was measured at -30 mV when the membrane was held at -90 mV and the L-type — current was measured at +20 mV when the membrane was held at -40 mV.
- the currents were measured twice at each concentration of mibefradil with 2 mm m between measurements.
- the dose dependent inhibition of T-type Ca 2+ current is shown m Fig. 3A.
- the 50% inhibitory concentration (IC 50 ) was 865 nM. No time-dependent inhibition was observed.
- mibefradil may permeate through the cell membrane to the cytoplasm and be trapped mside cells.
- the presence of mibefradil was examined m cells pre-mcubated with 20 ⁇ M of mibefradil using mass spectrometry. After 3 washes, mibefradil (peaked at 496 MW) was still detected m cells (Fig. 6B) .
- the localization of mibefradil m cells was examined by measuring the concentration of mibefradil m the pellets and supernatants after lysis of the cells.
- Insulin-dependent diabetes mellitus is characterized by the selective destruction of pancreatic ⁇ -cells.
- LVA Ca 2 * current m mouse ⁇ -cells Chronic treatment with cytokmes induced a low voltage- activated (LVA) Ca 2 * current m mouse ⁇ -cells.
- Exposure to cytokmes did not affect the profile of Ca 2* currents or basal [Ca 2 *], m glucagon-secreting ⁇ -cells.
- An increased Ca 2 * signal through LVA Ca 2 * channels may thus be a key feature m cytokine- induced ⁇ -cell destruction.
- a glucagon-secretmg cell line ( ⁇ - TC1) was also examined. This cell line, like ⁇ -cells, is more resistant to the cytotoxic effect of cytokmes.
- Treatment of ⁇ -TCl cells with IL-l ⁇ and IFN ⁇ failed to induce LVA Ca 2+ currents and did not alter the current density (Figs. 23C and 23D) . Therefore, the induction of LVA Ca 2+ currents and increased Ca 2+ current density observed after chronic treatment with cytokmes showed specificity for ⁇ -cells. LVA Ca 2 * channels are activated at low membrane potentials.
- cytokmes induce apoptosis m human pancreatic islet cells. Apoptosis is also the mode of cell death m the development of IDDM m the NOD mouse and m multiple low dose streptozotocm-mduced IDDM m the mouse, and is involved in ⁇ -cell destruction. As a marker of apoptosis, DNA fragmentation has been reported to precede ⁇ -cell lysis.
- ⁇ -TC3 cells a mouse ⁇ -cell line, were used to demonstrate the role of LVA Ca 2* channels in cytokine- ⁇ mediated DNA fragmentation. The LVA Ca 2 * current density was first examined before and after cytokine treatment.
- cytokines 25 U/ml IL-l ⁇ , 100 U/ml IFN ⁇ , and 100 U/ml TNF ⁇
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WO2001062740A1 (en) * | 2000-02-25 | 2001-08-30 | South Alabama Medical Science Foundation | Mibefradil analogues and their use |
WO2001062741A1 (en) * | 2000-02-25 | 2001-08-30 | South Alabama Medical Science Foundation | Tetrahydronaphtalene derivatives and their use |
US7157461B2 (en) | 2003-07-23 | 2007-01-02 | Bristol-Myers Squibb Co. | Substituted dihydropyrimidine inhibitors of calcium channel function |
US7166603B2 (en) | 2003-07-23 | 2007-01-23 | Bristol-Myers Squibb Co. | Dihydropyrimidone inhibitors of calcium channel function |
US7504431B2 (en) | 2004-04-16 | 2009-03-17 | Bristol-Myers Squibb Company | Sulfonyl amide inhibitors of calcium channel function |
EP2542155A2 (en) * | 2010-03-01 | 2013-01-09 | TAU Therapeutics LLC | Cancer diagnosis and imaging |
US9402848B2 (en) | 2008-02-29 | 2016-08-02 | Vm Therapeutics Llc | Method for treating pain syndrome and other disorders |
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Also Published As
Publication number | Publication date |
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EP1108068A1 (en) | 2001-06-20 |
EP1108068A4 (en) | 2002-12-04 |
JP2002525077A (en) | 2002-08-13 |
WO2000015845A9 (en) | 2001-12-13 |
AU6021799A (en) | 2000-04-03 |
CA2340586A1 (en) | 2000-03-23 |
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