CA2145214A1 - Agents for pre-symptomatic diagnosis, prevention and cure huntington's disease (hd) in humans - Google Patents
Agents for pre-symptomatic diagnosis, prevention and cure huntington's disease (hd) in humansInfo
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- CA2145214A1 CA2145214A1 CA 2145214 CA2145214A CA2145214A1 CA 2145214 A1 CA2145214 A1 CA 2145214A1 CA 2145214 CA2145214 CA 2145214 CA 2145214 A CA2145214 A CA 2145214A CA 2145214 A1 CA2145214 A1 CA 2145214A1
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Abstract
Anti sense nucleic acid and protein molecules, against which reagents can be prepared for accurate pre-symptomatic diagnostic, prevention and cure of Huntington's Disease in humans, are revealed .
Description
~1 45, ~14 Huntington' s Disease (HD) is an autosomal fll nAnt progressive and fatal neurodegenerative disease which starts in humans around age 40; it affects .0196 of the European population and about 1:8,000 people world wide, and sometimes, although rarely, occurs in its most seriouE
form, which includes muscle rijidity, in juveniles. The disease is characterised by irregular involuntary, choeric muscle contractions, cognitive and psychiatric disturbances and intellectual decline. There is no cure for the disease which progresses to death within 15-20 years from the date of onset (Bruyn, R.~.M. et al., J.
Neuroloq. Sci 95 :29-38 (1990) ) . The gene affected in HD
has been mapped to the short arm of chromosome 4 (Gusella, J.F. et al., Nature 306:234-238 (1983); Wexler, N.S. et al ., Nature 326 :194-19 7 (1984) ) . A mRNA transcript (IT15) cnnt~;n;ng a trinucleotide repeat (CAG) that is Pl~r~nA~A on a novel genel "huntingtin", located in the chromosomes of HD victims, was det~rm;n-~ to be the genetic basis of the disease (F~llnt;nqton~s Disease Collaborative Resear~h ~roul~, Cell 72:971-983 (1993) ) .
Further, it has been shown that de novo expansion of a (CAG)= is also involved in sporadic HD (Myers R.H. et al.
Nature Genetics 5:168-173 1993); that intermediate alleles, with respect to size of repeat, occur in people which do not show symptoms of the disease (Goldberg P Y.
et al 5 :174 -179 1993 ) and that the size of the repeat influences the rate of progression, age of onset and severity of the disease symptoms (Tanaka H., et al., Ann.
Neruol . 3 6: 63 0 - 6 3 5 19 9 4 ) .
Nevertheless, intensive investigation of the huntingtin gene product (protein), which is widely expressed in humans, has failed to show a biochemical relationship between the protein and the pathophysiological symptoms of Huntington' s disease. The latter involve ac, 1 ~t i nn of 5 a l ~
high quantities of lactic acid (lactic acidosiff) and premature loss of neurons in the brain, particularly in the caudate nucleus, putamen, globus pallidus, substantia nigra and occipital cortex (Perry, T. L . et al ., J .
Neurolog. Sci. 78 :139-150 (1987) ) . It has al30 been established that neither the expanded repeat nor the lack of the IT15 has a direct effect on the pathophysiology of HD which has lead to the conclusion that the ~D product ~huntingtin" gains an unknown function (Ambrose C.M. et al ., Somat. Cell Mol . Genet 20 :Z7-38 1994) . In any event, the unknown function of huntingtin protein must include the capability of influencing metabolic pathways in the human striatum, especially putamen and caudata, which lead to severe lactic acidosis in the neuron population of these tissues.
Results from a number of studies have suggested that faulty glucose me~hnl; ~m in specific populations of neurons is a factor in the neuropathological symptoms and general etiology of HD: e.g., (i) using "Positron Emission Tl ,ld~hy" disruption of glucose metabolism in the striatum of subjects generally at risk for ~D, was found to be the first sign of active HD before any clinical symptom waf3 apparent (Antonini. A. et al., Neurology 43:693 S (19g3); (ii) NMR spectroscopic analaysis of CSF and serum metabolic prof iles of ~D
patients revealed significant increase (6096) in pyruvate rr/nr~ ration in HD patients (Nicoli, ~., et al. Neurosci.
Lett. 154 (1-2): 47-51 (1993) ), and (iii) glucose transporters GLUT 3 and GLUT 1 activities are reduced in the cordate and putamen of late stage ~ID brains (Gambarino W.C. and Brennan W.A. et al ., J. Neruochem., 63 :1392-1397 (1994). Brain and liver metobalism is in many ways similar (~evor T.K., Biochime. 76:111-120 (1994); glycogen concentration was found to decreace in reaction to brain stimulation and increase when brain functions were stimulated and regional variations in normal brain glucose ~1~5~14 metabolic values (Wang G.J. et al., J. Nucl. Med 35:1459- = ~ =
1466 1994) . Also of particular interest to uncopuling of oxidative phosphorylation i~ adrenergic receptor mRNA and receptor uncoupling protein mRNA was identified in deep white fat tissue indicating that brown fat specific genes are expressed in adults and could mediate T3 induce thermogenesis (Krief et al. J. Clin. Invest. 91:344-349 1993 ) . The above results and, the involvement of localised neuronal lactic acidosis in the etiolog of HD, rasised the possibility that disruption of an important glucose metabolic parthway might be the maj or cause of the clinical symptoms of HD. -DESCRIPTION OF TE E EMBODIMENTS OF TEIE INVENTION
~ovel genes within the huntingtin disease locus:
We have discovered two genes, HuntL and HuntL4 in antisense arrangement to the sense strand of IT15 on the huntingtin gene (f igure la) . Transcription of the two genes is powered by 3 promoter systems (two with correlated TATA element, GC box and cap site, and one ~:
with TATA box and cap site) located in the 5'upstream region of the anti sense gene (figure lg) . The protein coding region of HuntL4 ( f igure lf ) spans IT15 exon including the CAG repeat and the f lanking CCG repeat, on the complementary strand of huntingtin gene. The protein cQdin~ region of ~untL (~igure lb) opens 50 bp 5 ' to the CAG repeat and overlaps 99 bases of HuntL4 (figure la) . The biQchemical characteristics of the protein products of the HuntL4 (figure lf) and HuntlI (figure ld) genes, which are described below, suggest that either of these proteins, can cause the clinical symptoms of HD, and their rnmh;nPA action might cause more severe symptoms of the disease. ~untL4 rnnti~inl:l the complementary sequence to the unstable, P~;~n~ hl e, trinucleotide repeat region demonstrated in IT15 to be ~1 45,~1~
the genetic cause of HD; therefore, the sequence in HuntL4 also expands in XD victims. In contrast to huntingtin, the protein encoded by IT15, HuntL4 has the biochemical characteristics required to cause the full disease phenotype. The genetic and physiological characteristics predicates HuntL4 as the actual cause o~ :
HD .
THE TeCTTT-T~C OF THE lNV~
(i) XuntL SEQ ID:NO:1 (Preddie, R.E. and Bergmann, J.E., Can. patent appl. #2,087,738 & 2,094,169; U.S.A. appl.
#08/050/578, 08/168,261; 08/215,683); (Bergmann JE. &
Preddie R.E. PCT appl.EP 9~3/03721; EU appI. 94914374.7) .
encodes encodes a novel tachykinin related neuropeptide pHuntH (SEQ ID:NO:2), figure 2b & 2c. The novel 33 amino acid neuropeptide can be phosphorylated at Ser 14 and can be further modified by post translation amidation o~ _ Leu 30, in specific tissue, to form XuntH (SEQ ID:~0:3) with the release of a dipeptide Arg-Arg. A secretory signal which can cleave the peptide between residues 31/32 will also provide an Arg-Arg dipeptide. pHuntH has a potential mitochrondial transit signal between amino acid 2 - 31.
HuntL4 (SEQ:ID:NO 4) contains 23 CTG and 10 CGG repeats.
It encodes a novel 211 amino acid. tachykinin-like neuropeptide, figure 2a & 2c. The polypeptide contains ~
serveral functionally important domains (figure lf) ~=
including a classical mito~l~rc n~l; 4l transit sequence (aa 2-60). A potential amidation site located at aa 62 will - ~
release a dipeptide, Arg-Arg~N2, in tissues where the d~ Liate amidation enzyme is active. It can be phosphorylated at ser 177. The domain of XuntL4 encoded by the CTG repeat region is part of a alpha helix in the centre of the protein. The alpha helical region includes 23 leu amino acids residues which forms the major part ~ 1 4 5 t ~ l~
oL a trans- membrane helix (26 aa), and four overlapping "leucine zippers". Trinucleotide expansion which leads to addition of leu residues in the tr~n~ dlle alpha helical region of Huntl4 is likely to have profound structural and functional consequences for the protein.
The severity of such consequences will be directly related to the number o~ additional Leu residues, e.g., the addition of 16 Leu residues will provide a second tl~n ,L, lle spanning helix in the polypeptide chain (the genetic cause of "achondroplasia" is thought to be a t~tlnn in the triln~ rane helix coding region of a gene) . The domain of HuntL4 encoded by the CGG repeat which flanks the CTG repeat encodes a highly basic region (10 arg residues) which can ~unction as a nuclear transit sequence, perhaps in a tissue specif ic manner The protein has potential secretory signals between ~ -amino acids 121/122; I24/125 and 127/128. l:rse of any of the latter sites will release an active neuropeptide oi~
90/87/84 amino acids. A degenerate "KH" like RNA binding domain in HuntL4 (amino acids is shown in figure 3a. The putative biochemical properties of HuntH and HuntL4 =.
which relate to HD are shown in Table #1. How the neuropeptides might cause the pathophysiological symptoms of HD are shown in the model n~ltl ;n~l in figure ~--#4a & 4b.
PHYSIOLOaTa~T. ROLE OF HUNTL4 and HUNTH IN TEE
NEI~ROPATHOLOGY OF HD.
As shown in figure 2a-c HuntH and HuntL4 are related to =~
tachykinins and to opioid type neuropeptides.
Tachykinins are highly active peptides which excite neurons, evoke behavioral responses, are potent vasodilators and secretagogues and contact directly or indirectly many smooth muscle cells (Nakajima et al., J.
Biol . Chem., 267:2437-2442 (1992) ) . Brain opioid neuropeptides are known to modulate the expression of ,QI 4s,~1q classical neurotransmitters in neurons (Weisskopf et al., Nature 362: 423-427 (1993)) .
Administration of synthetic (-2 mg of 99 . 9896 pure) amidated HuntH to rats caused neurotoxic lesions to develop in the rat striatum. The neurotoxic effect was enhanced when HuntX was injected in 50~ DMSO which facilicated penetration of the peptlde into cells. MK- ~=
801 blocked the formation of the lesions in response to EluntH administration. In other experiments MRI lactate imaging seemed to show that HuntH had an ef f ect on energy metabolism in mitochondria in rat brains.
Intrestingly, it was found that the formation of neurotoxic lesions in the rat striatum was duplicated when ~2 mg glucogen was administered in place of HuntH.
The presence of a region specific (Campos R:V., et al., Endocrinology 134: 2156-2164 (1994) ) glucagon sensitive, brain regulator system (Seto K,, et al., Exp. Clin.
Endocrinol. 93:69-72 (1993)) which tr~n~ rG~ the same glucagon receptor species found to mediate the effect of glucagon in rat liver, kidney, skeletal muscle, (Wheeler M. B., et al ., Endocrinology 133 :57-62 (1993) ), suggested to us that the neurotoxic effect of high levels of glucagon observed in rat striatum might have involved a receptor triggered response to the extrodinary high level of glucagon . We inf erred f rom the latter that receptor triggering was also a possible response in rat striatum to high levels of HuntX. This possibility provided a clue to how HuntH might affect neurons in brains and muscles o~ XD victims.
We used our proprietary computer assisted protein fingerprinting analysis technology to fingerprint XuntH
and HuntL4 using public data bases. These analyses allowed us to deduce that the neuropeptides could =
influence caterh~ minf~ (epinepherine and ~ ~145 ~14 norepinepherine) transduced glycogenolysis in two possible ways. One is by inhibiting the ~-~t~rhnl o~
methyltransferase, the enzyme which deactivates the catP~hnl i~m; nf~s, the other by activatiing adrenergic receptors in the plasma membrane. Transduction of adrenergic receptors by catchecolamines initiates the reactions that lead to the production of ATP from glycogen (glycogenolysis) in skeletal muscle, and glucose from glycogen in liver.
In addition, the fingerprinting results (table 1) indicated that HuntH and HuntL4 can affect activity of a number of other enzymes which are crucial for glycogenolysis and energy me~ilhnl; c:m Discovery of the enzymes and other proteins which can be inf luenced by HuntL4 and HuntH, occurrence of lactic acidosis in specific populations of neurons in HD
brains, knowledge that glucose metabolism is disturbed in asymptomatic HD, and our assumption that varying levels of glycogenolysis occur i~ different populations of brain neurons (brain and the adrenal mr~ r where catecholamines are produced have the same embryonic origin) made it possible to construct a simplistic bio~h~mi~1 model the disease.
The model shown in the embodiment of f igure 4a and 4b indicates how HuntL4 and HuntH concurrently ~ctivate synthesis and breakdown of glycogen in selected brain and muscle neurons. This is different to the normal glycogenolysis during which glycogen synthesis is inhibited while glycogen breakdown i8 avtivated, which leads to the production o~ large quantities of ATP
required by muscle contraction. Under the in~luence of HuntL4 and HuntH no ATP for use by muscle is produced.
5,~14 HuntL4, modified by a significant increase in the number of leu residues, distorts the plasma membrane in proportion to the number of leu residues added through trinucleotide expansion, possibly resulting in displ ~r~m~nt and inactivation or spurious activation of speci~ic membrane receptors or other functional proteins . The lose of glucose ~ransporters in the plasma membrane of HD brain neurons may be one result of HuntL4 induced plasma membrane distortion. In addition to distorting the membrane the leu composition of the transmembrane domain in wild type HuntL4 may allow the neuropeptide to occupy trans membrane sites of a number of other proteins (see figure 3a) and hence to modulate or mimic tr;3n~fllT~; ng~enzymatic activity of the proteins inVO1Yed~ PLUYLCLLI.._d modulation (down regulation) of specif ic membrane resident proteins involved in energy production may be a normal function of wild type HuntL4 in humans age 40+.
There is an interesting parallel in the epiderminology of non insulin fl~p~nflf~nT- diabetes mellitus (~IDDM) and ~ r; n~rhrine tran8duces alpha adrenergic receptors in liver, in the glycolytic pathway, which leads to synthesis o~ glucose. I~ rAnfllOfl l~untL4, is expressed in liver the ef~ect might be constant production oE
glucose by glycogenolysis. EluntL4 with limited trinucleotide expansion (i.e. expansions below the level which causes HD might affect the level of insulin receptors in the liver as appears to be the case of the glucose transporter in ~rain neurons.
It should be also noted that HuntL4 might modulate the atrionatriuetic Factor (ANF) localized in heart tissue.
If HuntL4 is expressed in heart tissue it can have an effect on the generation o~ angiotensins and hence on 4 ~, ~ 14 hypertension .
Given that HuntL4 and HuntH are not usually expressed in humans at detectable levels and, when expressed, are --likely to occur in body fluids, i.e., blood and CSF, presymptomatic test based on antibodies made to (CAGt~ P~R~(~PRRPA) and (rr,RRRT.RT~TRRR) from the c-terminal alld central part of HuntL4 respectively, and (VSPARQSPBASGR) from the n-tPrm;n~l o~ HuntH, have been developed for their detection and therapeutic methods aimed at preventing synthesis and blocking expression of the neuropeptides are being developed.
~ ~14~ 14 lv SEQ ID: NO :1 TCGCC ATG GCG GTC TCC CGC CCG GCA CGG CAG =:
TCC CCG GAG GCC TCG GGC CGA CTC GCG
GCG CCG CTC AGC ACC GGG GCA ATG AAT
GGG GCT CTG GGC CGC AGG TAA
5EQ ID: NO:2 MAVSRPARQSPEASGRLA~PLSTGAMNGALGRR
SEQ ID: NO: 3 MAvsRpARQspEAsGRlAApLsTGAMNGAL-~H2 45,a~14 SEQ ID:NO:4 ll TCCCTC ATG GGC TCT GGG TTG CTG GGT CAC TCT GTC TCT CCG GGG CCG
GGG GTT CGT GTC GCC GGC . CCC AGG CTG CAG GGT TAC CGC CAT CCC CGC
CGT AGC CTG GGA CCG CCG GGA CAG GGA GCT GCA GCG GGC CCA AAC TCA
CGG TCG GTG C~G CGG CTC CTC AGC CAC TGC CGG GCC GGG TGG CGG C
GGG CGG CGG CGG GGG CGC CTG CGG CTG AGG CAG CAG CGG CTT GCC TGC
GGC GGC GGC TGA CGG AAG CTG AGG AGG CGG CGG CGG CGG CGG CGG CGG
TGG CGG CTG TTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG
CTG CTG CTG CTG CTG CTG CTG CTG CTG GAA GGA CTT GAG GGA CTC GAA
GGC CTA CAT CAG CTT TTC CAG GGT CGC CAT GGC GGT CAC CCG CCC GGC
ACG GCA GTC CCC GGA GGC CTC GGC CCC ACT CGC GGC GCC GCT CAG CAC
CGG GGC AAT GAA TGG GGC TCT GGG CCG CAG GTA A~A GCA GAA CCT GAG
CGG CCG TCC ATC TTG GAC CCG TCC CGG CAG CCC CCA CGG CGC CTT GCG
TCC CAG ACG TGC GCC G~C GGA GGC GGG GCC GCG CCG GCG GAG GCG GGG
CCA CGC CGG CCA GCA TGA TTG
SEQ ID:NOS
M G S G L L G H S V S P G P G V R V A G P R L Q G Y R H P R R S
L G P P G Q G A A A G P N S R S V Q R L L S H C R A G W R R G R
R R G R L R L R Q Q R L C L R R R L R E L R R R R R R R R R W R
L L L L L L L L L L L L L L L L L L L L L L L E G L B G L R G L
H Q L F Q G R H G G H P P G T A V P G G L G P T R G A A Q H R G
N E W G S G P Q v X A E P E R P S I L D P S R Q P P R R L A S Q
T C A G G G G A A P A E A G P R R P A
~ 5~
SEQ ID:NO:6 1~
TCAAACGCCTG~At'.A(~l"AA-'TTAGGCTTAGATGAGCAGATCTCTTAGGTGGTGCTTAAAA
Nl~ llAcTA~ Af~c(~TGATt~T~ AA~TAAATGAhATGTccTGAcAATGTAGGAAGAG
f'ArAAATCTCAGG~ l 1 lAAGTGccAcTAfl~AAAA~AAATcTccc~TTGGAcGcGc CCTCACTCCCCNGCCCCCCCCCTGCCCCGCCACTCCCGCCGAGGCTN~ TAAAAt~.C~TTA
TGTcATccAcTGrt~ Af~ATcTcTccAGcTct~AAAGf~ Lc~L~ ~AAGTGTccc GCGCCCCCAGCC(~ r.~('TA(~A~ GGGAaGTGGCACTGAGCaAATCTCG
GCTCCTCCAA(3~ 'ACaACGCAGTTAAAA~AArCCCCGCCCTG~l l ~ l~CAAA
TAAaATt~AAAr~Af.'TTCTCG~f~r~'A~TCTTCCCATCCTG~ Ar~.N~.CGAGANGGCTGA
GGCCGTGACTCCCCAGCCTGAATTCAGGAC,AGG~ 'AAA(~AAf~N~TCTGt~ ~C~'TCG
CGAGAGt'-At'AAr~-AA~ ~AA(~-TGAGGGAGCGGCCGTGAAGTGGGGGAAGGCCTCGCC
CAGGAG~n ~ C~ l ~ ATG
~45,~L~
M~TABO~IC ENZYMES LIRELY TO BE~ ACTIVATED/INACTIVATED
BY H~JTL4 AND HU~H
HUNTI,4 J~U~TH
, rob bly prob~bly probubly prob~bl upreh~ulntos downr~gul~t~ uprogulat~ t-.
~Y-lA/2A adrenergic c~techol-o-methyl- ~-2A adrenergic pyruvato receptor tr~ns~erase receptor precursor (1 hydrolipo~~~d~ cy~
ein er~n~r~ omp ~ent) phosphata~ e Ca'~ calmodium sennitive ;~denyl cyclase hormone senditive ,~-adrenergic triacylglycerol receptor Ca'~ c;llmodium senYiitive adenyl cyclase up or down rogulAtou up or dcwn rogulcto~
A~P ~ynthase protein 1 dihydropyridine sensitive I-type 3keletal muscle c~lcium channel ateri~l natriur~tic poptide receptor A Coenzyme PQQ
dihydropyridine sen~itive ~-type skeletal muocle calcium channel tyrosine protein kinaoe receptor c~lcium channel B, and B, aynaptic veoicle ~minc transporter ~) ~5, ~14 1~
Figure la Location on HuntL4 and HuntL with respect to IT15 in the huntingtin gene region on human chromosome #4 Figure lb =SEQ ID:NO:l The protein coding region of HuntL
Figure lc =SEQ ID:NO:2 Deduced amino acid sequence of HuntH
Figure ld =SEQ ID: NO: 3 Sequence of synthetic amidated HuntH
Figure le =SEQ ID: NO: 4 Coding region of HuntL4 gene Figure lf Deduced amino acid sequence of HuntL4 = mitochondrial transit signal (classical ! ) NH~ = potential amidation site ~ = secretory signal (in HuntH & HuntL4) LLLL . . . L = amino acids encoded by (CTG) = repeat region ,R.~ = potential nuclear transit signal = trans membrane alpha helix L....L = Leucine zipper '= strong alpha helix region Figure lg ~ 'r L re~
5' up-stream region o~ HuntL4 gene the region ~ nti~ins two correlated (TATA element-GCC box-cap site) promoter systems and one (TATA element- cap site) promoter system .
= GC box ~- TATA element ~~ = cap site ~1 ~sa/~
Symbol s:
ep epinephrine norep norepinephrine COMT catechol o-methyltran8ferase cY-adren.rec ~-adrenergic receptor ,B-adren.rec ,~-adrenergic receptor IP3 inositol 1,4,5-tripho8phate PPP phosphoprotein phosphatase GSase glycogen synthase GPase glycogen phophorylase glu glucose G-1-P glucose-1-phosphate G - 6 - P gluco se - 6 - phosphate G-6-Pase glucose-6-phosphatase pyr pyruvate lac lactate lac . a lactic acid triglc triglycerides hstglase hormone sensitive triacylglycerol lipase uncpl ox.phos uncoupling of oxidative phosphorylation
form, which includes muscle rijidity, in juveniles. The disease is characterised by irregular involuntary, choeric muscle contractions, cognitive and psychiatric disturbances and intellectual decline. There is no cure for the disease which progresses to death within 15-20 years from the date of onset (Bruyn, R.~.M. et al., J.
Neuroloq. Sci 95 :29-38 (1990) ) . The gene affected in HD
has been mapped to the short arm of chromosome 4 (Gusella, J.F. et al., Nature 306:234-238 (1983); Wexler, N.S. et al ., Nature 326 :194-19 7 (1984) ) . A mRNA transcript (IT15) cnnt~;n;ng a trinucleotide repeat (CAG) that is Pl~r~nA~A on a novel genel "huntingtin", located in the chromosomes of HD victims, was det~rm;n-~ to be the genetic basis of the disease (F~llnt;nqton~s Disease Collaborative Resear~h ~roul~, Cell 72:971-983 (1993) ) .
Further, it has been shown that de novo expansion of a (CAG)= is also involved in sporadic HD (Myers R.H. et al.
Nature Genetics 5:168-173 1993); that intermediate alleles, with respect to size of repeat, occur in people which do not show symptoms of the disease (Goldberg P Y.
et al 5 :174 -179 1993 ) and that the size of the repeat influences the rate of progression, age of onset and severity of the disease symptoms (Tanaka H., et al., Ann.
Neruol . 3 6: 63 0 - 6 3 5 19 9 4 ) .
Nevertheless, intensive investigation of the huntingtin gene product (protein), which is widely expressed in humans, has failed to show a biochemical relationship between the protein and the pathophysiological symptoms of Huntington' s disease. The latter involve ac, 1 ~t i nn of 5 a l ~
high quantities of lactic acid (lactic acidosiff) and premature loss of neurons in the brain, particularly in the caudate nucleus, putamen, globus pallidus, substantia nigra and occipital cortex (Perry, T. L . et al ., J .
Neurolog. Sci. 78 :139-150 (1987) ) . It has al30 been established that neither the expanded repeat nor the lack of the IT15 has a direct effect on the pathophysiology of HD which has lead to the conclusion that the ~D product ~huntingtin" gains an unknown function (Ambrose C.M. et al ., Somat. Cell Mol . Genet 20 :Z7-38 1994) . In any event, the unknown function of huntingtin protein must include the capability of influencing metabolic pathways in the human striatum, especially putamen and caudata, which lead to severe lactic acidosis in the neuron population of these tissues.
Results from a number of studies have suggested that faulty glucose me~hnl; ~m in specific populations of neurons is a factor in the neuropathological symptoms and general etiology of HD: e.g., (i) using "Positron Emission Tl ,ld~hy" disruption of glucose metabolism in the striatum of subjects generally at risk for ~D, was found to be the first sign of active HD before any clinical symptom waf3 apparent (Antonini. A. et al., Neurology 43:693 S (19g3); (ii) NMR spectroscopic analaysis of CSF and serum metabolic prof iles of ~D
patients revealed significant increase (6096) in pyruvate rr/nr~ ration in HD patients (Nicoli, ~., et al. Neurosci.
Lett. 154 (1-2): 47-51 (1993) ), and (iii) glucose transporters GLUT 3 and GLUT 1 activities are reduced in the cordate and putamen of late stage ~ID brains (Gambarino W.C. and Brennan W.A. et al ., J. Neruochem., 63 :1392-1397 (1994). Brain and liver metobalism is in many ways similar (~evor T.K., Biochime. 76:111-120 (1994); glycogen concentration was found to decreace in reaction to brain stimulation and increase when brain functions were stimulated and regional variations in normal brain glucose ~1~5~14 metabolic values (Wang G.J. et al., J. Nucl. Med 35:1459- = ~ =
1466 1994) . Also of particular interest to uncopuling of oxidative phosphorylation i~ adrenergic receptor mRNA and receptor uncoupling protein mRNA was identified in deep white fat tissue indicating that brown fat specific genes are expressed in adults and could mediate T3 induce thermogenesis (Krief et al. J. Clin. Invest. 91:344-349 1993 ) . The above results and, the involvement of localised neuronal lactic acidosis in the etiolog of HD, rasised the possibility that disruption of an important glucose metabolic parthway might be the maj or cause of the clinical symptoms of HD. -DESCRIPTION OF TE E EMBODIMENTS OF TEIE INVENTION
~ovel genes within the huntingtin disease locus:
We have discovered two genes, HuntL and HuntL4 in antisense arrangement to the sense strand of IT15 on the huntingtin gene (f igure la) . Transcription of the two genes is powered by 3 promoter systems (two with correlated TATA element, GC box and cap site, and one ~:
with TATA box and cap site) located in the 5'upstream region of the anti sense gene (figure lg) . The protein coding region of HuntL4 ( f igure lf ) spans IT15 exon including the CAG repeat and the f lanking CCG repeat, on the complementary strand of huntingtin gene. The protein cQdin~ region of ~untL (~igure lb) opens 50 bp 5 ' to the CAG repeat and overlaps 99 bases of HuntL4 (figure la) . The biQchemical characteristics of the protein products of the HuntL4 (figure lf) and HuntlI (figure ld) genes, which are described below, suggest that either of these proteins, can cause the clinical symptoms of HD, and their rnmh;nPA action might cause more severe symptoms of the disease. ~untL4 rnnti~inl:l the complementary sequence to the unstable, P~;~n~ hl e, trinucleotide repeat region demonstrated in IT15 to be ~1 45,~1~
the genetic cause of HD; therefore, the sequence in HuntL4 also expands in XD victims. In contrast to huntingtin, the protein encoded by IT15, HuntL4 has the biochemical characteristics required to cause the full disease phenotype. The genetic and physiological characteristics predicates HuntL4 as the actual cause o~ :
HD .
THE TeCTTT-T~C OF THE lNV~
(i) XuntL SEQ ID:NO:1 (Preddie, R.E. and Bergmann, J.E., Can. patent appl. #2,087,738 & 2,094,169; U.S.A. appl.
#08/050/578, 08/168,261; 08/215,683); (Bergmann JE. &
Preddie R.E. PCT appl.EP 9~3/03721; EU appI. 94914374.7) .
encodes encodes a novel tachykinin related neuropeptide pHuntH (SEQ ID:NO:2), figure 2b & 2c. The novel 33 amino acid neuropeptide can be phosphorylated at Ser 14 and can be further modified by post translation amidation o~ _ Leu 30, in specific tissue, to form XuntH (SEQ ID:~0:3) with the release of a dipeptide Arg-Arg. A secretory signal which can cleave the peptide between residues 31/32 will also provide an Arg-Arg dipeptide. pHuntH has a potential mitochrondial transit signal between amino acid 2 - 31.
HuntL4 (SEQ:ID:NO 4) contains 23 CTG and 10 CGG repeats.
It encodes a novel 211 amino acid. tachykinin-like neuropeptide, figure 2a & 2c. The polypeptide contains ~
serveral functionally important domains (figure lf) ~=
including a classical mito~l~rc n~l; 4l transit sequence (aa 2-60). A potential amidation site located at aa 62 will - ~
release a dipeptide, Arg-Arg~N2, in tissues where the d~ Liate amidation enzyme is active. It can be phosphorylated at ser 177. The domain of XuntL4 encoded by the CTG repeat region is part of a alpha helix in the centre of the protein. The alpha helical region includes 23 leu amino acids residues which forms the major part ~ 1 4 5 t ~ l~
oL a trans- membrane helix (26 aa), and four overlapping "leucine zippers". Trinucleotide expansion which leads to addition of leu residues in the tr~n~ dlle alpha helical region of Huntl4 is likely to have profound structural and functional consequences for the protein.
The severity of such consequences will be directly related to the number o~ additional Leu residues, e.g., the addition of 16 Leu residues will provide a second tl~n ,L, lle spanning helix in the polypeptide chain (the genetic cause of "achondroplasia" is thought to be a t~tlnn in the triln~ rane helix coding region of a gene) . The domain of HuntL4 encoded by the CGG repeat which flanks the CTG repeat encodes a highly basic region (10 arg residues) which can ~unction as a nuclear transit sequence, perhaps in a tissue specif ic manner The protein has potential secretory signals between ~ -amino acids 121/122; I24/125 and 127/128. l:rse of any of the latter sites will release an active neuropeptide oi~
90/87/84 amino acids. A degenerate "KH" like RNA binding domain in HuntL4 (amino acids is shown in figure 3a. The putative biochemical properties of HuntH and HuntL4 =.
which relate to HD are shown in Table #1. How the neuropeptides might cause the pathophysiological symptoms of HD are shown in the model n~ltl ;n~l in figure ~--#4a & 4b.
PHYSIOLOaTa~T. ROLE OF HUNTL4 and HUNTH IN TEE
NEI~ROPATHOLOGY OF HD.
As shown in figure 2a-c HuntH and HuntL4 are related to =~
tachykinins and to opioid type neuropeptides.
Tachykinins are highly active peptides which excite neurons, evoke behavioral responses, are potent vasodilators and secretagogues and contact directly or indirectly many smooth muscle cells (Nakajima et al., J.
Biol . Chem., 267:2437-2442 (1992) ) . Brain opioid neuropeptides are known to modulate the expression of ,QI 4s,~1q classical neurotransmitters in neurons (Weisskopf et al., Nature 362: 423-427 (1993)) .
Administration of synthetic (-2 mg of 99 . 9896 pure) amidated HuntH to rats caused neurotoxic lesions to develop in the rat striatum. The neurotoxic effect was enhanced when HuntX was injected in 50~ DMSO which facilicated penetration of the peptlde into cells. MK- ~=
801 blocked the formation of the lesions in response to EluntH administration. In other experiments MRI lactate imaging seemed to show that HuntH had an ef f ect on energy metabolism in mitochondria in rat brains.
Intrestingly, it was found that the formation of neurotoxic lesions in the rat striatum was duplicated when ~2 mg glucogen was administered in place of HuntH.
The presence of a region specific (Campos R:V., et al., Endocrinology 134: 2156-2164 (1994) ) glucagon sensitive, brain regulator system (Seto K,, et al., Exp. Clin.
Endocrinol. 93:69-72 (1993)) which tr~n~ rG~ the same glucagon receptor species found to mediate the effect of glucagon in rat liver, kidney, skeletal muscle, (Wheeler M. B., et al ., Endocrinology 133 :57-62 (1993) ), suggested to us that the neurotoxic effect of high levels of glucagon observed in rat striatum might have involved a receptor triggered response to the extrodinary high level of glucagon . We inf erred f rom the latter that receptor triggering was also a possible response in rat striatum to high levels of HuntX. This possibility provided a clue to how HuntH might affect neurons in brains and muscles o~ XD victims.
We used our proprietary computer assisted protein fingerprinting analysis technology to fingerprint XuntH
and HuntL4 using public data bases. These analyses allowed us to deduce that the neuropeptides could =
influence caterh~ minf~ (epinepherine and ~ ~145 ~14 norepinepherine) transduced glycogenolysis in two possible ways. One is by inhibiting the ~-~t~rhnl o~
methyltransferase, the enzyme which deactivates the catP~hnl i~m; nf~s, the other by activatiing adrenergic receptors in the plasma membrane. Transduction of adrenergic receptors by catchecolamines initiates the reactions that lead to the production of ATP from glycogen (glycogenolysis) in skeletal muscle, and glucose from glycogen in liver.
In addition, the fingerprinting results (table 1) indicated that HuntH and HuntL4 can affect activity of a number of other enzymes which are crucial for glycogenolysis and energy me~ilhnl; c:m Discovery of the enzymes and other proteins which can be inf luenced by HuntL4 and HuntH, occurrence of lactic acidosis in specific populations of neurons in HD
brains, knowledge that glucose metabolism is disturbed in asymptomatic HD, and our assumption that varying levels of glycogenolysis occur i~ different populations of brain neurons (brain and the adrenal mr~ r where catecholamines are produced have the same embryonic origin) made it possible to construct a simplistic bio~h~mi~1 model the disease.
The model shown in the embodiment of f igure 4a and 4b indicates how HuntL4 and HuntH concurrently ~ctivate synthesis and breakdown of glycogen in selected brain and muscle neurons. This is different to the normal glycogenolysis during which glycogen synthesis is inhibited while glycogen breakdown i8 avtivated, which leads to the production o~ large quantities of ATP
required by muscle contraction. Under the in~luence of HuntL4 and HuntH no ATP for use by muscle is produced.
5,~14 HuntL4, modified by a significant increase in the number of leu residues, distorts the plasma membrane in proportion to the number of leu residues added through trinucleotide expansion, possibly resulting in displ ~r~m~nt and inactivation or spurious activation of speci~ic membrane receptors or other functional proteins . The lose of glucose ~ransporters in the plasma membrane of HD brain neurons may be one result of HuntL4 induced plasma membrane distortion. In addition to distorting the membrane the leu composition of the transmembrane domain in wild type HuntL4 may allow the neuropeptide to occupy trans membrane sites of a number of other proteins (see figure 3a) and hence to modulate or mimic tr;3n~fllT~; ng~enzymatic activity of the proteins inVO1Yed~ PLUYLCLLI.._d modulation (down regulation) of specif ic membrane resident proteins involved in energy production may be a normal function of wild type HuntL4 in humans age 40+.
There is an interesting parallel in the epiderminology of non insulin fl~p~nflf~nT- diabetes mellitus (~IDDM) and ~ r; n~rhrine tran8duces alpha adrenergic receptors in liver, in the glycolytic pathway, which leads to synthesis o~ glucose. I~ rAnfllOfl l~untL4, is expressed in liver the ef~ect might be constant production oE
glucose by glycogenolysis. EluntL4 with limited trinucleotide expansion (i.e. expansions below the level which causes HD might affect the level of insulin receptors in the liver as appears to be the case of the glucose transporter in ~rain neurons.
It should be also noted that HuntL4 might modulate the atrionatriuetic Factor (ANF) localized in heart tissue.
If HuntL4 is expressed in heart tissue it can have an effect on the generation o~ angiotensins and hence on 4 ~, ~ 14 hypertension .
Given that HuntL4 and HuntH are not usually expressed in humans at detectable levels and, when expressed, are --likely to occur in body fluids, i.e., blood and CSF, presymptomatic test based on antibodies made to (CAGt~ P~R~(~PRRPA) and (rr,RRRT.RT~TRRR) from the c-terminal alld central part of HuntL4 respectively, and (VSPARQSPBASGR) from the n-tPrm;n~l o~ HuntH, have been developed for their detection and therapeutic methods aimed at preventing synthesis and blocking expression of the neuropeptides are being developed.
~ ~14~ 14 lv SEQ ID: NO :1 TCGCC ATG GCG GTC TCC CGC CCG GCA CGG CAG =:
TCC CCG GAG GCC TCG GGC CGA CTC GCG
GCG CCG CTC AGC ACC GGG GCA ATG AAT
GGG GCT CTG GGC CGC AGG TAA
5EQ ID: NO:2 MAVSRPARQSPEASGRLA~PLSTGAMNGALGRR
SEQ ID: NO: 3 MAvsRpARQspEAsGRlAApLsTGAMNGAL-~H2 45,a~14 SEQ ID:NO:4 ll TCCCTC ATG GGC TCT GGG TTG CTG GGT CAC TCT GTC TCT CCG GGG CCG
GGG GTT CGT GTC GCC GGC . CCC AGG CTG CAG GGT TAC CGC CAT CCC CGC
CGT AGC CTG GGA CCG CCG GGA CAG GGA GCT GCA GCG GGC CCA AAC TCA
CGG TCG GTG C~G CGG CTC CTC AGC CAC TGC CGG GCC GGG TGG CGG C
GGG CGG CGG CGG GGG CGC CTG CGG CTG AGG CAG CAG CGG CTT GCC TGC
GGC GGC GGC TGA CGG AAG CTG AGG AGG CGG CGG CGG CGG CGG CGG CGG
TGG CGG CTG TTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG CTG
CTG CTG CTG CTG CTG CTG CTG CTG CTG GAA GGA CTT GAG GGA CTC GAA
GGC CTA CAT CAG CTT TTC CAG GGT CGC CAT GGC GGT CAC CCG CCC GGC
ACG GCA GTC CCC GGA GGC CTC GGC CCC ACT CGC GGC GCC GCT CAG CAC
CGG GGC AAT GAA TGG GGC TCT GGG CCG CAG GTA A~A GCA GAA CCT GAG
CGG CCG TCC ATC TTG GAC CCG TCC CGG CAG CCC CCA CGG CGC CTT GCG
TCC CAG ACG TGC GCC G~C GGA GGC GGG GCC GCG CCG GCG GAG GCG GGG
CCA CGC CGG CCA GCA TGA TTG
SEQ ID:NOS
M G S G L L G H S V S P G P G V R V A G P R L Q G Y R H P R R S
L G P P G Q G A A A G P N S R S V Q R L L S H C R A G W R R G R
R R G R L R L R Q Q R L C L R R R L R E L R R R R R R R R R W R
L L L L L L L L L L L L L L L L L L L L L L L E G L B G L R G L
H Q L F Q G R H G G H P P G T A V P G G L G P T R G A A Q H R G
N E W G S G P Q v X A E P E R P S I L D P S R Q P P R R L A S Q
T C A G G G G A A P A E A G P R R P A
~ 5~
SEQ ID:NO:6 1~
TCAAACGCCTG~At'.A(~l"AA-'TTAGGCTTAGATGAGCAGATCTCTTAGGTGGTGCTTAAAA
Nl~ llAcTA~ Af~c(~TGATt~T~ AA~TAAATGAhATGTccTGAcAATGTAGGAAGAG
f'ArAAATCTCAGG~ l 1 lAAGTGccAcTAfl~AAAA~AAATcTccc~TTGGAcGcGc CCTCACTCCCCNGCCCCCCCCCTGCCCCGCCACTCCCGCCGAGGCTN~ TAAAAt~.C~TTA
TGTcATccAcTGrt~ Af~ATcTcTccAGcTct~AAAGf~ Lc~L~ ~AAGTGTccc GCGCCCCCAGCC(~ r.~('TA(~A~ GGGAaGTGGCACTGAGCaAATCTCG
GCTCCTCCAA(3~ 'ACaACGCAGTTAAAA~AArCCCCGCCCTG~l l ~ l~CAAA
TAAaATt~AAAr~Af.'TTCTCG~f~r~'A~TCTTCCCATCCTG~ Ar~.N~.CGAGANGGCTGA
GGCCGTGACTCCCCAGCCTGAATTCAGGAC,AGG~ 'AAA(~AAf~N~TCTGt~ ~C~'TCG
CGAGAGt'-At'AAr~-AA~ ~AA(~-TGAGGGAGCGGCCGTGAAGTGGGGGAAGGCCTCGCC
CAGGAG~n ~ C~ l ~ ATG
~45,~L~
M~TABO~IC ENZYMES LIRELY TO BE~ ACTIVATED/INACTIVATED
BY H~JTL4 AND HU~H
HUNTI,4 J~U~TH
, rob bly prob~bly probubly prob~bl upreh~ulntos downr~gul~t~ uprogulat~ t-.
~Y-lA/2A adrenergic c~techol-o-methyl- ~-2A adrenergic pyruvato receptor tr~ns~erase receptor precursor (1 hydrolipo~~~d~ cy~
ein er~n~r~ omp ~ent) phosphata~ e Ca'~ calmodium sennitive ;~denyl cyclase hormone senditive ,~-adrenergic triacylglycerol receptor Ca'~ c;llmodium senYiitive adenyl cyclase up or down rogulAtou up or dcwn rogulcto~
A~P ~ynthase protein 1 dihydropyridine sensitive I-type 3keletal muscle c~lcium channel ateri~l natriur~tic poptide receptor A Coenzyme PQQ
dihydropyridine sen~itive ~-type skeletal muocle calcium channel tyrosine protein kinaoe receptor c~lcium channel B, and B, aynaptic veoicle ~minc transporter ~) ~5, ~14 1~
Figure la Location on HuntL4 and HuntL with respect to IT15 in the huntingtin gene region on human chromosome #4 Figure lb =SEQ ID:NO:l The protein coding region of HuntL
Figure lc =SEQ ID:NO:2 Deduced amino acid sequence of HuntH
Figure ld =SEQ ID: NO: 3 Sequence of synthetic amidated HuntH
Figure le =SEQ ID: NO: 4 Coding region of HuntL4 gene Figure lf Deduced amino acid sequence of HuntL4 = mitochondrial transit signal (classical ! ) NH~ = potential amidation site ~ = secretory signal (in HuntH & HuntL4) LLLL . . . L = amino acids encoded by (CTG) = repeat region ,R.~ = potential nuclear transit signal = trans membrane alpha helix L....L = Leucine zipper '= strong alpha helix region Figure lg ~ 'r L re~
5' up-stream region o~ HuntL4 gene the region ~ nti~ins two correlated (TATA element-GCC box-cap site) promoter systems and one (TATA element- cap site) promoter system .
= GC box ~- TATA element ~~ = cap site ~1 ~sa/~
Symbol s:
ep epinephrine norep norepinephrine COMT catechol o-methyltran8ferase cY-adren.rec ~-adrenergic receptor ,B-adren.rec ,~-adrenergic receptor IP3 inositol 1,4,5-tripho8phate PPP phosphoprotein phosphatase GSase glycogen synthase GPase glycogen phophorylase glu glucose G-1-P glucose-1-phosphate G - 6 - P gluco se - 6 - phosphate G-6-Pase glucose-6-phosphatase pyr pyruvate lac lactate lac . a lactic acid triglc triglycerides hstglase hormone sensitive triacylglycerol lipase uncpl ox.phos uncoupling of oxidative phosphorylation
Claims (21)
1. A nucleic acid molecule substantially free of natural contaminants selected from the group Huntl and HuntL4 wherein the said sequences are SEQ ID NO:1 and SEQ ID NO:4 respectively.
2. A protein product substantially free of natural contaminants selected from the group pHuntH, HuntH and HuntL4 wherein said sequences are SEQ ID NO: 2, SEQ ID NO: 3 and SEQ ID NO: 5 respectively .
3. A protein product of claim 2 which is encoded by a nucleic acid molecule of claim 1
4. A nucleic acid molecule, HuntLreg, substantially free of natural contaminants wherein the said sequence is SEQ ID NO:6.
5. The chemically or biologically altered forms of a nucleic acid molecule selected from the molecules of claim 1 and claim 4.
6. The chemically or biologically altered form of a protein selected from the group of proteins of claim 2.
7. The use of the nucleic acid molecules from the group of claim 4 to program the expression of genes in any natural or artificial biological system.
8. Ribozymes and other RNA cleaving molecules produced from nucleic acid molecules from the group of claim 2.
9. An anti sense nucleic acid molecule designed to prevent the activity of the promoter elements in the nucleic acid molecule from claim 4.
10. An anti sense nucleic acid molecule designed to specifically detect the presence of nucleic acid molecules and mutated nucleic acid molecules from the group of claim 1.
11. An anti sense nucleic acid molecule designed to prevent expression of proteins from the nucleic acid molecules from the group of claim 1
12. A antibody designed to detect the presence of a protein from the group of claim 2.
13. An anti peptide reagent designed to detect the presence of a protein from the group of claim 2 .
14. A antibody designed and "humanised" for blocking the activity of a protein from the group of claim 2 .
15 . A method for treating Huntington's Disease which comprises administration to an individual an effective amount of an inhibitor of a nucleic acid molecule from the group of claim 1 or an inhibitor of a protein from the group of claim 2 or an inhibitor of transcription regulatory sequence from the group of claim 4.
16. A method for treating the disease in claim 15 which comprises administration to an individual an inhibitor of the alpha adrenergic receptor or epinephrine or norepinephrine.
17. A method of Gene Therapy for treating the diseases in 15 which comprises replacing a nucleic acid molecule from the group of claim 1.
18. A method of Gene Therapy for treating the disease in claim 15 which comprises replacing a nucleic acid in claim 4 .
19 A method of claim 15 for treating Non Insulin Dependent Diabetes Mellitus and Hypertension.
20. A method of claim 17 for treating the diseases of claim 19.
21. A method of claim 18 for treating the diseases of claim 19.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2145214 CA2145214A1 (en) | 1995-02-28 | 1995-02-28 | Agents for pre-symptomatic diagnosis, prevention and cure huntington's disease (hd) in humans |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA 2145214 CA2145214A1 (en) | 1995-02-28 | 1995-02-28 | Agents for pre-symptomatic diagnosis, prevention and cure huntington's disease (hd) in humans |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2145214A1 true CA2145214A1 (en) | 1996-08-29 |
Family
ID=4155484
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA 2145214 Abandoned CA2145214A1 (en) | 1995-02-28 | 1995-02-28 | Agents for pre-symptomatic diagnosis, prevention and cure huntington's disease (hd) in humans |
Country Status (1)
| Country | Link |
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| CA (1) | CA2145214A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US8969299B2 (en) | 2011-06-08 | 2015-03-03 | Kai Pharmaceuticals, Inc. | Therapeutic agents for regulating serum phosphorus |
| US8987200B2 (en) | 2006-11-16 | 2015-03-24 | Kai Pharmaceuticals, Inc. | Polycationic calcium modulator peptides for the treatment of hyperparathyroidism and hypercalcemic disorders |
| US8999932B2 (en) | 2009-07-29 | 2015-04-07 | Kai Pharmaceuticals, Inc. | Therapeutic agents for reducing parathyroid hormone levels |
-
1995
- 1995-02-28 CA CA 2145214 patent/CA2145214A1/en not_active Abandoned
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|---|---|---|---|---|
| US8987200B2 (en) | 2006-11-16 | 2015-03-24 | Kai Pharmaceuticals, Inc. | Polycationic calcium modulator peptides for the treatment of hyperparathyroidism and hypercalcemic disorders |
| US8999932B2 (en) | 2009-07-29 | 2015-04-07 | Kai Pharmaceuticals, Inc. | Therapeutic agents for reducing parathyroid hormone levels |
| US9278995B2 (en) | 2009-07-29 | 2016-03-08 | Kai Pharmaceuticals, Inc. | Therapeutic agents for reducing parathyroid hormone levels |
| US9567370B2 (en) | 2009-07-29 | 2017-02-14 | Kai Pharmaceuticals, Inc. | Therapeutic agents for reducing parathyroid hormone levels |
| US9701712B2 (en) | 2009-07-29 | 2017-07-11 | Kai Pharmaceuticals, Inc. | Therapeutic agents for reducing parathyroid hormone levels |
| US10280198B2 (en) | 2009-07-29 | 2019-05-07 | Kai Pharmaceuticals, Inc. | Therapeutic agents for reducing parathyroid hormone levels |
| US8969299B2 (en) | 2011-06-08 | 2015-03-03 | Kai Pharmaceuticals, Inc. | Therapeutic agents for regulating serum phosphorus |
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