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  • C12N2740/16043—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• the disclosure relates generally to molecules, including polypeptides, that alter the subcellular location of PDE11A4, and methods of using such compounds as treatments for disease.
• the present disclosure provides a novel class of inhibitors of PDEl lA4e.g, a useful therapeutic target for treating diseases and disorders including, but not limited to, social deficits associated with schizophrenia, bipolar disorder, or autism and cognitive deficits/dementia associated with age-related cognitive decline, traumatic brain injury, or Alzheimer’s disease.
• the disclosure provides an isolated fragment of PDE1 1 A4 comprising a GAF-B binding sequence and comprising or consisting of a polypeptide sequence of SEQ ID NO: 1, or fragments thereof having from at least about 70% to at least about 99% identity thereto.
• the disclosure provides an isolated fragment of PDE11 A4 comprising a GAF-B binding sequence and comprising or consisting of a polypeptide sequence of SEQ ID NO: 2, or fragments thereof having from at least about 70% to at least about 99% identity thereto.
• SEQ ID NO: 2 is similar to SEQ ID NO: 1, but contains a 14 AA spacer (SEQ ID NO: 4) on its N-terminus.
• the disclosure provides a polynucleotide encoding an isolated fragment of the invention.
• the present disclosure provides vectors and host cells for preparing a polynucleotide of the invention using recombinant methods.
• the host cell is a mammalian cell (e.g., CHO cell, a HEK- 293 cell, or an Sp2.0 cell).
• compositions comprising an isolated fragment of the invention and a physiologically compatible carrier medium.
• a pharmaceutical composition provides a therapeutically effective amount for the treatment or prevention of a disease or disorder alleviated by inhibiting PDE11 A4 activity e.g. in a patient in need thereof.
• the disease or disorder is associated with cognitive decline.
• the disease or disorder is dementia, Alzheimer’s Disease (AD) including mild Alzheimer's disease and early-onset Alzheimer’s disease, Down’s syndrome, vascular dementia (cerebral amyloid angiopathy and stroke), dementia with Lewy bodies, HIV dementia, Mild Cognitive Impairment (MCI); Age- Associated Memory Impairment (AAMI); Age-Related Cognitive Decline (ARCD) (including age-related cognitive decline of associative long-term memories (aLTMs), dementia associated with traumatic brain injury, preclinical Alzheimer's Disease (PCAD); Cognitive Impairment No Dementia (CIND), and cognitive decline associate with spatial memory , other depression-related behaviors, additional anxiety-related behaviors, sensorimotor gating, or social behaviors.
• a method comprises administering to the patient a therapeutically effective amount of an isolated fragment of the invention.
• a method comprises administering to the patient a therapeutically effective amount of a pharmaceutical composition the invention.
• an isolated fragment of the invention is administered in a dosage unit form.
• the dosage unit comprises a physiologically compatible carrier medium.
• a method treats or prevents a disease or disorder is associated with cognitive decline.
• the disease or disorder is selected from dementia, Alzheimer’s Disease (AD) including mild Alzheimer's disease and early-onset Alzheimer’s disease, Down’s syndrome, vascular dementia (cerebral amyloid angiopathy and stroke), dementia with Lewy bodies, HIV dementia, Mild Cognitive Impairment (MCI); Age-Associated Memory Impairment (AAMI); Age-Related Cognitive Decline (ARCD) (including age-related cognitive decline of associative long-term memories (aLTMs), dementia associated with traumatic brain injury, preclinical Alzheimer's Disease (PCAD); Cognitive Impairment No Dementia (CIND), and cognitive decline associate with spatial memory, other depression-related behaviors, additional anxiety-related behaviors, sensorimotor gating, and social behaviors.
• Fig 1 is an image showing how PDE11 A4 mRNA expression is restricted to the HIPP. Protein is expressed in neuronal cell bodies, dendrites, and axons.
• Figs. 2A-2C illustrate increases in hippocampal PDE11 A expression.
• Fig. 2A is a graph of experimental data show ing how hippocampal PDE11 A expression is increased in old vs. young mice.
• Fig. 2B. is a graph of experimental data showing how hippocampal PDE11A expression is increased in adult (18-40yrs) vs. prenatal humans.
• Fig. 2C is a graph of experimental data showing how hippocampal PDE11 A expression is increased in demented vs. non-demented aged humans (>75yrs) with a history of TBI. Post hoc *vs. Young, prenatal, or ‘No’ group, P ⁇ 0.05.
• A.U. arbitrary units.
• FIGs. 3A-3C illustrate aging preferentially impairs aLTMs in mice.
• Fig. 4A is a graph of experimental data showing male and female old PDE11 A WT mice (WT-0) having no memory for STFP 7 days after training; however old PDE11 A KO mice (KO-O) and heterozygous mice show robust memory equivalent to that of young (Y) mice.
• Fig. 5A is an image of non-limiting example that due to the large size of PDE11 A4 (>3kb), a lentivirus was used to overexpress a GFP-mPDEllA4 fusion or GFP alone (negative control).
• Fig. 5B shows western blots of DHIPP and VHIPP that illustrate titrating virus delivery allows overexpression of PDE11A4 in a dorsal ⁇ ventral gradient, as is seen in vivo.
• Fig. 5D is a graph of experimental data illustrating mice trained on STFP and tested 7 days after training.
• KO mice expressing GFP in the hippocampus show strong aLTM for STFP; however, KO mice overexpressing PDE11A4 in the hippocampus (that is, mimicking the state of an “old WT”) fail to show significant aLTM for STFP.
• Fig. 5E is a graph of experimental data illustrating the ability of PDE11 A4 overexpression to impair aLTM, as KO mice treated with either the GFP or PDE1 1 A4 lentivirus show strong rLTM for NSOR.
• FIGs. 6A-6K illustrate that PDE11 A homodimerization reverses molecular/biochemical phenotypes associated with aging independently of SI 62 phosphorylation.
• H(2) 16.34, PO.OOl
• SI 62 i.e., S162A
• Fig. 7A is an image and graph of experimental data illustrating phosphomimic mutations ofPDEHA4 at serines 117 and 124 (117D124D) synergize, increasing the accumulation of PDE11A4 in distinct structures.
• Phosphoresistant mutations S117AS124A have the opposite effect.
• Fig. 7B is a graph and image showing how biochemical fractionation of S117DS124 shifts PDE11A4 from the cytosol to the membrane.
• Fig. 7C is a graph of experimental data and image showing how SI 17 and S124 also synergize at the level of phosphorylation.
• Fig. 7E is a graph of experimental data normalizing the hyperaccumulation that is seen with SI 17D124D.
• Fig. 7F is a graph of experimental data and image showing expression of SI 17D124D mimics age-related decreases in cGMP levels.
• Fig. 7G is a graph of experimental data and image showing how expression of the isolated GAF-B domain has the opposite effect as what is shown in Fig. 7F.
• Fig 8. is an image showing immuno-fluorescence with a total PDE11A antibody (top — green) and pS117/pS124-PDEHA4 antibody (bottom — green) suggest that age-related increases in PDE11A expression occur in a compartment-specific manner. KOs virally overexpressing PDE11AWT show the same pattern as “old” .
• Fig 9 is a graph of experimental data and image showing age-related increases in VHIPP PDE11 A4 protein expression occurs preferentially in membrane fractions, consistent with the in vitro studies showing S117D/S124D mutations shift PDE11A4 from the cytosol to the membrane.
• Fig 10 is an image showing how mPdel la4-mCherry reports transcriptional activity in ventral CAI (vCAl ) but not ventral dentate gyrus (vDG), consistent with endogenous expression pattern of mPdellA4 (see also Fig. 1).
• vCAl ventral CAI
• vDG ventral dentate gyrus
• the ubiquitous Pgk-mCherry construct reports transcription in both subregions.
• Fig. 12A is an image showing widefield fluorescence’s ability to label PDE11 A4 mRNA (red) using RNAscope.
• a confocal microscope in the USC 1RF are used to collect z- stack images.
• Fig. 12B is an image showing an example of subcellular resolution provided by confocal microscope. Shown is PDE11A4 protein (green) in COS1 cells counterstained for the nuclear marker DAPI (blue). Optical sections through the cell (shown to the right and bottom) clarify whether labeling observed from above is in the nucleus or cytosol.
• Figs. 13A-13G illustrate that disruption PDE11 A homodimerization in vivo selectively decreases PDE11A4 expression in a compartment-specific manner and is sufficient to decrease PDE11 A4 accumulations in ghost axons that occur with age.
• Fig. 13A is an image of a lentiviral construct containing either mCherry (i.e., negative control) or an mCherry -tagged isolated GAF-B domain (GAF-B) was injected bilaterally into dorsal and ventral CAI of hippocampus.
• Fig. 13B is an image showing that that injection of the GAF-B construct disrupted PDE11A4 homodimerization; whereas, mCherry alone did not.
• Fig. 13C is an image showing stereotaxic delivery' of the lentiviral constructs (mCherry and mCherry - GAF-B) targeting dorsal (DHIPP) and ventral (VHIPP) CAI sub-regions of the hippocampus resulted in high expression within CAI in all subjects, with a subset of mice demonstrating a diffuse expression in dentate gyrus, CA2, and CA3.
• Fig. 13C is an image showing stereotaxic delivery' of the lentiviral constructs (mCherry and mCherry - GAF-B) targeting dorsal (DHIPP) and ventral (VHIPP) CAI sub-regions of the hippocampus resulted in high expression within CAI in all subjects, with a subset of mice demonstrating a diffuse expression in dentate gyrus, CA2, and CA3.
• Fig. 13C is an image showing stereotaxic delivery' of the lentiviral constructs (mCherry and mCher
• FIG. 13D is an image showing stereotaxic delivery of the lentiviral constructs (mCherry and mCherry-GAF-B) targeting dorsal (DHIPP) and ventral (VHIPP) CAI sub-regions of the hippocampus resulted in high expression within CAI in all subjects, with a subset of mice demonstrating a diffuse expression in dentate gyms, CA2, and CA3.
• Fig 13E is an image showing expression of mCherry-GAF-B decreased PDE11 A4 expression in distal dendrites (dotted-arrows) and axons (solid arrows).
• Fig 13F is an image showing the GAF-B construct was also able to reduce age-related increases in so-called “PDEl lA ghost axons” (i.e., filamentous structures where PDE11A4 accumulates with age; shown: “140/150” cocktail of antibodies).
• PDE lA ghost axons i.e., filamentous structures where PDE11A4 accumulates with age; shown: “140/150” cocktail of antibodies.
• Figs. 14A-14G illustrate that disrupting PDE11 A homodimerization in the hippocampus of old mice is sufficient to reverse age-related decline of remote long-term social memory. Fig.
• SOR social odor recognition training
• administer refers to (1) providing, giving, dosing, and/or prescribing by either a health practitioner or his authorized agent or under his or her direction according to the disclosure; and/or (2) putting into, taking or consuming by the mammal, according to the disclosure.
• co-administration encompass administration of two or more active pharmaceutical ingredients to a subject so that both active pharmaceutical ingredients and/or their metabolites are present in the subject at the same time.
• Co-administration includes simultaneous administration in separate compositions, administration at different times in separate compositions, or administration in a composition in which two or more active pharmaceutical ingredients are present. Simultaneous administration in separate compositions and administration in a composition in which both agents are present are preferred.
• active pharmaceutical ingredient and “drug” include the polypeptides, polynucleotides, and compositions described herein.
• active pharmaceutical ingredient and “drug” may also include those compounds described herein that bind PDE11A4 and thereby modulate (e.g. inhibit) PDE11A4 activity.
• the term in vivo refers to an event that takes place in a subject s body.
• zn vitro refers to an event that takes places outside of a subject’s body.
• In vitro assays encompass cell-based assays in which cells alive or dead are employed and may also encompass a cell-free assay in which no intact cells are employed.
• the term “effective amount” or “therapeutically effective amount” refers to that amount of a compound or combination of compounds as described herein that is sufficient to effect the intended application including, but not limited to, disease treatment.
• a therapeutically effective amount may vary depending upon the intended application (in vitro or in vivo), or the subject and disease condition being treated (e.g, the weight, age and gender of the subject), the severity of the disease condition, the manner of administration, etc., which can readily be determined by one of ordinary skill in the art.
• the term also applies to a dose that will induce a particular response in target cells (e.g, the reduction of platelet adhesion and/or cell migration).
• the specific dose will vary depending on the particular compounds chosen, the dosing regimen to be followed, whether the compound is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which the compound is carried.
• a prophylactic effect includes delaying or eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.
• the terms “treat,” “treatment,” and/or “treating” may refer to the management of a disease, disorder, or pathological condition, or symptom thereof with the intent to cure, ameliorate, stabilize, and/or control the disease, disorder, pathological condition or symptom thereof.
• control may include the absence of condition progression, as assessed by the response to the methods recited herein, where such response may be complete (e.g, placing the disease in remission) or partial (e.g., lessening or ameliorating any symptoms associated with the condition).
• the terms “prevent,” “preventing,” and/or “prevention” may refer to reducing the risk of developing a disease, disorder, or pathological condition.
• the terms “modulate” and “modulation” refer to a change in biological activity for a biological molecule (e.g, a protein, gene, peptide, antibody, and the like), where such change may relate to an increase in biological activity (e.g, increased activity, agonism, activation, expression, upregulation, and/or increased expression) or decrease in biological activity (e.g, decreased activity, antagonism, suppression, deactivation, downregulation, and/or decreased expression) for the biological molecule.
• the compounds described herein may modulate (e.g., inhibit) PDE11A4 protein.
• the compounds described herein may selectively modulate (e.g, selectively inhibit) PDE11A4 protein as compared to other PDE11A proteins. In some embodiments, the compounds described herein may selectively modulate (e.g., selectively inhibit) PDE11A4 protein as compared to other PDE or PDE11 A proteins.
• Modulate” and “modulation” also include changing the subcellular localization and/or location of PDE11A4. “Modulate” and “modulation” also include disrupting and/or preventing homodimerization of PDE11A4. “Modulate” and “modulation” also include direct modulation of PDE11A4 (e.g. modulation of catalytic activity ofPDEHA4).
• Modulate” and “modulation” also include indirect modulation of PDE11A4 (e.g. disrupting and/or preventing homodimerization of PDE1 1 A4).
• Inhibit” and “inhibiting” also include changing the subcellular localization and/or location of PDE11A4.
• “Inhibit” and “inhibiting” also include disrupting and/or preventing homodimerization of PDE11 A4.
• “Inhibit” and “inhibiting” also include direct inhibition of PDE11A4 (e.g. inhibition of catalytic activity ofPDEHA4).
• Inhibit” and “inhibiting” also include indirect inhibition of PDE11A4 (e.g. disrupting and/or preventing homodimerization of PDE11A4 and/or degrading PDE11A4).
• the terms “QD,” “qd,” or “q.d.” mean quaque die, once a day, or once daily.
• the terms “BID,” “bid,” or “b.i.d.” mean bis in die, twice a day, or twice daily.
• the terms “TID,” “tid,” or “t.i.d.” mean ter in die, three times a day, or three times daily.
• the terms “Q1D,” “qid,” or “q.i.d ” mean quater in die, four times a day, or four times daily.
• conservatively modified variants refers to those nucleic acids which encode identical or essentially identical amino acid sequences, or where the nucleic acid does not encode an amino acid sequence, to essentially identical sequences. Because of the degeneracy of the genetic code, a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide.
• nucleic acid variations are “silent variations,” which are one species of conservatively modified variations. Every nucleic acid sequence herein which encodes a polypeptide also describes every possible silent variation of the nucleic acid.
• each codon in a nucleic acid except AUG, which is ordinarily the only codon for methionine, and TGG, which is ordinarily the only codon for tryptophan
• TGG which is ordinarily the only codon for tryptophan
• “conservatively modified variants” include individual substitutions, deletions or additions to a polypeptide sequence which result in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles of the disclosure.
• the following eight groups contain amino acids that are conservative substitutions for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), V aline (V); 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C), Methionine (M)(see, e.g., Creighton, Proteins (1984)).
• the term is a conservative substitution for one another: 1) Alanine (A), Glycine (G); 2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (
• “conservative sequence modifications” are used to refer to amino acid modifications that do not significantly affect or alter the binding characteristics of the antibody containing the amino acid sequence.
• percent identical in the context of two or more nucleic acids or polypeptide sequences, refers to two or more sequences or subsequences that are the same.
• Two sequences are “substantially identical” if two sequences have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or, when not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
• the identity exists over a region that is at least about 50 nucleotides (or 10 amino acids) in length, or more preferably over a region that is 100 to 500 or 1000 or more nucleotides (or 20, 50, 200 or more amino acids) in length.
• sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
• test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary', and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
• sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
• a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
• Methods of alignment of sequences for companson are well known in the art.
• Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482c (1970), by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol.
• BLAST and BLAST 2.0 algorithms Two examples of algorithms that are suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al., Nuc. Acids Res. 25:3389-3402, 1977; and Altschul et al., J. Mol. Biol. 215:403-410, 1990, respectively.
• Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold (Altschul et al., supra).
• initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
• the word hits are extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always ⁇ 0). For ammo acid sequences, a sconng matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
• the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
• the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873- 5787, 1993).
• One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
• P(N) the smallest sum probability
• Lor example a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
• the percent identity between two amino acid sequences can also be determined using the algorithm of E. Meyers and W. Miller, Comput. Appl. Biosci. 4: 11-17, 1988) which has been incorporated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
• the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch, J. Mol. Biol.
• nucleic acid is used herein interchangeably with the term “polynucleotide” and refers to deoxyribonucleotides or ribonucleotides and polymers thereof in either single- or double-stranded form.
• the term encompasses nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides.
• Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNAs).
• nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions) and complementary sequences, as well as the sequence explicitly indicated.
• degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed- base and/or deoxyinosine residues (Balzer et al., (1991) Nucleic Acid Res. 19:5081; Ohtsuka et al., (1985) J. Biol. Chem. 260:2605-2608: and Rossolini et al, (1994) Mol. Cell. Probes 8:91-98).
• operably linked in the context of nucleic acids refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, it refers to the functional relationship of a transcriptional regulatory sequence to a transcribed sequence.
• a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system.
• promoter transcriptional regulatory sequences that are operably linked to a transcribed sequence are physically contiguous to the transcribed sequence, i.e., they are cis-acting.
• some transcriptional regulatory sequences, such as enhancers need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.
• polypeptide and protein are used interchangeably herein to refer to a polymer of amino acid residues.
• the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring ammo acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses conservatively modified variants thereof.
• pharmaceutically acceptable salt refers to salts derived from a variety of organic and inorganic counter ions known in the art. Pharmaceutically acceptable acid addition salts can be formed with inorganic acids and organic acids.
• Preferred inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid and phosphoric acid.
• Preferred organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumanc acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p- toluenesulfonic acid and salicylic acid.
• Pharmaceutically acceptable base addition salts can be formed with inorganic and organic bases.
• Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese and aluminum.
• Organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines and basic ion exchange resins. Specific examples include isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine.
• the pharmaceutically acceptable base addition salt is chosen from ammonium, potassium, sodium, calcium, and magnesium salts.
• cocrystal refers to a molecular complex derived from a number of cocrystal formers known in the art. Unlike a salt, a cocrystal typically does not involve hydrogen transfer between the cocrystal and the drug, and instead involves intermolecular interactions, such as hydrogen bonding, aromatic ring stacking, or dispersive forces, between the cocrystal former and the drug in the crystal structure.
• “Pharmaceutically acceptable carrier” or “pharmaceutically acceptable excipient” or “physiologically compatible” carrier or carrier medium is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and inert ingredients.
• the use of such pharmaceutically acceptable carriers or pharmaceutically acceptable excipients for active pharmaceutical ingredients is well known in the art. Except insofar as any conventional pharmaceutically acceptable carrier or pharmaceutically acceptable excipient is incompatible with the active pharmaceutical ingredient, its use in the therapeutic compositions of the disclosure is contemplated. Additional active pharmaceutical ingredients, such as other drugs, can also be incorporated into the described compositions and methods.
• ranges are used herein to describe, for example, physical or chemical properties such as molecular weight or chemical formulae, all combinations and subcombinations of ranges and specific embodiments therein are intended to be included.
• Use of the term “about” when referring to a number or a numerical range means that the number or numerical range referred to is an approximation within experimental variability (or within statistical experimental error), and thus the number or numerical range may vary. The variation is typically from 0% to 15%, or from 0% to 10%, or from 0% to 5% of the stated number or numerical range.
• PDE11A phosphodiesterase 11 A
• PDE1 1 A is a member of the large phosphodiesterase enzyme family and was originally cloned in 2000.
• the enzyme is derived from a single gene product, hydrolyzes both cAMP and cGMP and exists in 4 isoforms.
• the enzyme is predominantly expressed in the brain.
• PDE11 A is found in the anterior hippocampus and more specifically in neurons in the superficial layer of CAI, the subiculum and the amygdalohippocampal area of the hippocampus.
• PDE11 A4 is the only PDE whose expression in brain emanates from the hippocampus, a region of the brain associated with associated long term memory (aLTM). Little is known of the signaling pathways lying up or downstream of PDE1 1 A4, but it has been shown that PDE11A appears to regulate important signals for memory consolidation, including glutamatergic and calcium/calmodulin-dependent kinase II (CamKII) signaling, as well as protein synthesis. It has been shown that cAMP and cGMP signaling are decreased in aged and demented hippocampus in rats and humans. These age- related decreases in cyclic nucleotides are associated with increased expression of PDE11A4 in rodents and in humans with hippocampal dementia, versus non-demented aged subjects with a history of traumatic brain injury.
• amKII calcium/calmodulin-dependent kinase II
• the PDE11 A family which breaks down cAMP and cGMP, is comprised of a single gene that is spliced into 4 isoforms, PDE11A1 through PDE11A4.
• the longest isoform, PDE11 A4 is the isoform that is expressed in brain and it is -95% homologous across mouse, rat and human. This high degree of homology argues that the results obtained in rodent models translates across species.
• PDE11A single nucleotide polymorphisms (SNPs) have been associated with major depressive disorder (MDD), suicide risk, antidepressant response in patients with MDD, and lithium response in patients with bipolar disorder. Both MDD and BPD have been conceptualized as diseases of accelerated aging.
• PDE11 A4 is expressed in the brain.
• PDE11 A4 was found in brain because PDE11 A knockout (KO) mice was phenotyped and what few phenotypes they had were found to be of relevance to ventral hippocampal function.
• the search for a PDEHA isoform in brain was directed to the ventral hippocampal formation (VHIPP) to detect expression restricted to this small brain region.
• VHIPP ventral hippocampal formation
• PDE11 A4 was strongly expressed in neurons of the superficial layer of CAI, the subiculum, and the adjacently connected amygdalohippocampal area (AHi) of the VHIPP (Figs.
• PDE11 A is a highly druggable enzyme — makes PDE11 A a very attractive drug target because it stands to selectively restore aberrant cyclic nucleotide signaling in a brain region affected by age- related decline without directly affecting signaling in other brain regions or peripheral organs that might lead to unwanted side effects.
• PDE11 A4 molecularly defines an exceptionally discrete circuit within a brain region key to learning and memory, making it ripe for the study of age-related cognitive decline.
• cAMP and cGMP signaling are decreased in the aged and demented hippocampus (rodents and humans), particularly when there is a history of traumatic brain injury (TBI).
• TBI traumatic brain injury
• PDE11 A KO mice Consistent with PDE1 lA4’s restricted expression pattern, PDE11 A KO mice appear normal on a wide range of sensory, motor and anxiety/depression-related behaviors, and show no gross peripheral pathology at least up to 1 year of age (later ages not assessed,). Instead, PDE11 A KO mice exhibit select social phenotypes such as preferring to interact with other PDE11 A KO mice vs wild-type (WT) mice and showing differences in the consolidation of social memories. PDE11A KO mice also have an increased sensitivity to the behavioral effects of lithium.
• PDE11A appears to regulate signals that are important for memory consolidation, including glutamatergic and calcium/calmodulin-dependent kinase II (CamKII) signaling as well as protein synthesis.
• PDEs are discretely localized to specific subcellular domains. As a result, PDEs do not simply control the total cellular content of cyclic nucleotides, they generate individual pools or nanodomains of cyclic nucleotide signaling. Such subcellular compartmentalization of cyclic nucleotide signaling allows a single cell to respond discretely to diverse intra- and extracellular signals. Thus, where a PDE is localized is just as important to its overall function as is its catalytic activity.
• cyclases and PDEs responsible for generating and breaking down cAMP/cGMP are expressed more in the cytosol than the membrane (like PDE11 A4), others are enriched in the membrane (like the closely related PDE2A and PDE10A).
• cyclic nucleotide signaling deficits observed in bipolar disorder and Alzheimer’s disease appear to be more prominent in the cytosolic as opposed to membrane fractions.
• Associative long-term memories (aLTMs) particularly those involving friends and family — are more susceptible to age-related cognitive decline than are recognition longterm memories (rLTMs) for reasons that are not well understood. The lack of knowledge of the molecular mechanisms that govern age-related decline slows the development of novel therapeutics.
• Age-related increases in phosphodiesterase 11 A (PDE11 A), an enzyme that breaks down cAMP/cGMP and regulates social behaviors, may be a fundamental mechanism underlying age-related cognitive decline of aLTMs for social experiences.
• PDE11 A phosphodiesterase 11 A
• the best controlled studies to date suggest that the longest isoform PDE11A4 is almost exclusively expressed in the ventral hippocampal formation (a k a. anterior HIPP in primates), specifically within neurons of the subiculum, superficial layer of CAI, and the adjacently connected amygdalohippocampal area. This makes PDE11A4 the ONLY PDE to be preferentially expressed in the HIPP, a brain region key to social aLTMs.
• PDEs are expressed more in the cytosol than the membrane (e.g., PDE11A), while others are more highly expressed in the membrane versus cytosol (including PDE2A, PDE9A and PDE10A.
• PDEs are localized to specific subcellular domains, they are able to regulate individual pools or nanodomains of cyclic nucleotide signaling.
• Such subcellular compartmentalization of cyclic nucleotide signaling allows a single cell to respond specifically to simultaneous intra- and/or extracellular signals. Therefore, the subcellular localization of any PDE is equally important to its actual catalytic activity when considering its function.
• PDEs can become overexpressed and/or mislocalized with age and/or disease, which compromises the integrity of this physiological segregation of signals. Indeed, age-related diseases and neuropsychiatric diseases can show a loss of cyclic nucleotide signaling in one subcellular compartment but not another, suggesting therapeutic strategies should optimally target enzymes in a compartment-specific manner.
• phosphodiesterase 11A (PDE11A) has garnered particular interest in the context of altered cyclic nucleotide signaling related to ARCD and early-onset Alzheimer's disease. PDE11A is encoded by a single gene and has four isoforms.
• While protein for PDE11 A4 the isoform expressed in brain — is found across all subcell ular compartments, it is particularly enriched in the cytosolic versus membrane and nuclear compartments.
• the PDE11 A catal tic domain is located within the C-terminal region, which is common to all isoforms, while the N-terminal region serves a regulatory function and is unique to each isoform.
• the regulatory N-terminus of PDE1 1 A4, the longest PDE11 A isoform, is unique in that it contains two full GAF (cGMP binding PDE, Anabaena adenylyl cyclase and E. coli FhlA) domains.
• the GAF-A domain binds cGMP as a potential allosteric regulatory site and the GAF-B domain regulates protein-protein interactions, including homodimerization.
• PDE11 A4 is unique in that it is the only PDE whose expression in brain emanates solely from the extended hippocampal formation, a brain region critical to learning and memory and vulnerable to age-related deficits in cyclic nucleotide signaling. Possibly contributing to these hippocampal cyclic nucleotide signaling deficits are age-related increases in PDE11 A4 expression that are conserved across mice, rats and humans.
• PDE11 A4 protein expression is deleterious as 1) Pdel la KO mice are protected against age-related cognitive decline (ARCD) of remote social associative longterm memories (aLTMs) and 2) mimicking age-related overexpression of PDE11A4 in the CAI field of hippocampus of either young or old Pdel la KO mice is sufficient to mimic ARCD of remote social aLTMs.
• ARCD age-related cognitive decline
• aLTMs remote social associative longterm memories
• disrupting PDE11A4 homodimerization in vivo may represent a therapeutic option capable of treating age-related increases in PDE11 A4 expression in a compartment-specific manner and, thus, ARCD of social memories.
• social preference of C57BL/6J mice can be altered by manipulating PDE11 A4 homodimerization selectively within the CAI field of hippocampus.
• inhibitors of PDE11A4 include molecules (e.g. polypeptides) capable of changing the subcellular localization and/or location of PDE11A4, and/or disrupting and/or preventing homodimerization of PDE11 A4, and/or degrading PDE11A4.
• PDE11 A4 is a useful therapeutic target for treating social deficits associated with schizophrenia, bipolar disorder, or autism as well as for treating cognitive deficits/dementia associated with age-related cognitive decline, traumatic bram injury', or Alzheimer’s disease.
• the disclosure provides an isolated fragment of PDE11A4, which comprises an isolated GAF-B domain of PDE11A4 that disrupts homodimerization and is capable of 1) altering social preferences/compatibility within the context of neuropsychiatric or neurodevelopmental disorders, 2) reversing cognitive decline associated with aging, dementia associated with traumatic brain injury, and/or Alzheimer’s disease, and/or 3) alleviating other disorders where PDE11A4 forms accumulated proteinopathies, particularly in the membrane fraction.
• the role of PDE11A4 in actual brain function is explored, and intramolecular mechanisms that control how PDE11 A4 functions in terms of enzymatic activity and subcellular trafficking are examined.
• the disclosure includes molecules capable of inhibiting PDE11 A4, including but not limited to disrupting and/or preventing homodimerization of PDE11A4, and including but not limited to isolated fragments of PDE11A4 comprising a GAF-B binding sequence.
• the PDE11A4 inhibitor is a molecule (e.g. polypeptide) capable of changing the subcellular localization and/or location of PDE11A4.
• the PDE11 A4 inhibitor is a molecule (e.g. polypeptide) capable of disrupting and/or preventing homodimerization of PDE11A4.
• the PDE11 A4 inhibitor is a molecule (e.g.
• the PDE11 A4 degraders of the disclosure disrupt and/or prevent homodimenzation of PDE11A4, thereby changing the subcellular localization and/or location of PDE11A4 and triggering degradation of PDE11A4.
• the PDE11 A4 inhibitor is a PDE11 A4 selective inhibitor.
• the PDE11A4 inhibitor of the disclosure is about 1-fold, about 5-fold, about 10-fold, about 20-fold, about 50-fold, about 100-fold, about 500-fold, or about 1000-fold more selective for PDE1 1 A4 over PDE1 1 Al , PDE1 1 A2, and/or PDE11 A3.
• the disclosure provides an isolated fragment of PDE11A4 comprising a GAF-B binding sequence. See for example Smith et al., Mol. Psychiatry 26:7107-7117 (2011), which is incorporated by reference herein in its entirety.
• the isolated fragment comprises or consists of a polypeptide sequence of SEQ ID NO: 1, or a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.
• the isolated fragment comprises or consists of a polypeptide sequence of SEQ ID NO: 2, or a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.
• the isolated fragment of PDE11A4 comprising a GAF-B binding sequence further comprises a fusion protein.
• the fusion protein comprises the isolated fragment and a moiety selected from the group consisting of an immunoglobulin fragment (e.g., an immunoglobulin Fc domain), serum albumin (e.g., human serum albumin), transferrin, and Fn3, or variants thereof.
• an immunoglobulin fragment e.g., an immunoglobulin Fc domain
• serum albumin e.g., human serum albumin
• transferrin e.g., transferrin, and Fn3, or variants thereof.
• the isolated fragment of PDE11 A4 comprising a GAF-B binding sequence comprises the isolated fragment conjugated to a non-protein polymer, such as polyethylene glycol.
• the isolated fragment of PDE11 A4 comprising a GAF-B binding sequence comprises the isolated fragment operably linked to an immunoglobulin Fc domain. In some embodiments, the isolated fragment of PDE11A4 comprising a GAF-B binding sequence comprises the isolated fragment operably linked to human serum albumin.
• the disclosure also provides polynucleotides encoding any of the polypeptides, including SEQ ID NO: 1 or 2, described herein. In aspects, the disclosure also provides a method of making any of the polynucleotides described herein. Polynucleotides can be made and expressed by procedures known in the art.
• sequence of a desired polypeptide and/or fragment thereof, and nucleic acid encoding such antibody, or fragment thereof can be determined using standard sequencing techniques.
• a nucleic acid sequence encoding a desired polypeptide and/or fragment thereof, may be inserted into various vectors (such as cloning and expression vectors) for recombinant production and characterization.
• the disclosure provides polynucleotides encoding the polypeptide sequence of SEQ ID NO: 1.
• the polynucleotide encoding the SEQ ID NO: 1 encodes an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.
• the disclosure provides polynucleotides encoding the polypeptide sequence of SEQ ID NO: 2.
• the polynucleotide encoding the SEQ ID NO: 2 encodes an amino acid sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.
• the disclosure provides the polynucleotide sequence of SEQ ID NO: 3, or a polynucleotide sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.
• SEQ ID NO: 1, SEQ ID NO: 2, and/or SEQ ID NO: 3 further comprise a tag (e g. mCherry).
• the disclosure provides a polynucleotide sequence comprising amino acids 561-567 of PDE11A and/or further comprising a tag (e.g. mCherry). (see SEQ ID NO: 5 for reference):
• polynucleotides of this disclosure can be obtained using chemical synthesis, recombinant methods, or PCR. Methods of chemical polynucleotide synthesis are well known in the art and need not be described in detail herein. One of skill in the art can use the sequences provided herein and a commercial DNA synthesizer to produce a desired DNA sequence.
• a polynucleotide comprising a desired sequence can be inserted into a suitable vector, and the vector in turn can be introduced into a suitable host cell for replication and amplification, as further discussed herein.
• Polynucleotides may be inserted into host cells by any means known in the art. Cells are transformed by introducing an exogenous polynucleotide by direct uptake, endocytosis, transfection, F-mating or electroporation. Once introduced, the exogenous polynucleotide can be maintained within the cell as a non-integrated vector (such as a plasmid) or integrated into the host cell genome.
• the polynucleotide so amplified can be isolated from the host cell by methods well known within the art. See, e.g., Sambrook et al., 1989.
• PCR allows reproduction of DNA sequences.
• PCR technology is well known in the art and is described in U.S. Patent Nos. 4,683,195, 4,800,159, 4,754,065 and 4,683,202, as well as PCR: The Polymerase Chain Reaction, Mullis et al., eds., Birkauswer Press, Boston, 1994.
• RNA can be obtained by using the isolated DNA in an appropriate vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can then be isolated using methods well known to those of skill in the art, as set forth in Sambrook et al., 1989, for example.
• a vector is selected that is optimized for expression of polypeptides in CHO or CHO-derived cells, or in NSO cells.
• Exemplary vectors are described, e.g., in Running Deer et al., Biotechnol. Prog. 20:880-889 (2004).
• Suitable cloning and expression vectors can include a variety of components, such as promoter, enhancer, and other transcriptional regulatory sequences.
• the vector may also be constructed to allow for subsequent cloning of polypeptide into different vectors.
• Suitable cloning vectors may be constructed according to standard techniques, or may be selected from a large number of cloning vectors available in the art. While the cloning vector selected may vary according to the host cell intended to be used, useful cloning vectors will generally have the ability to self-replicate, may possess a single target for a particular restriction endonuclease, and/or may carry genes for a marker that can be used in selecting clones containing the vector.
• Suitable examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mpl8, mpl9, pBR322, pMB9, ColEl, pCRl, RP4, phage DNAs, and shuttle vectors such as pSA3 and pAT28.
• Bluescript e.g., pBS SK+
• mpl8 mpl9 mpl9
• pBR322 mpl9
• ColEl ColEl
• pCRl pCRl
• RP4 phage DNAs
• shuttle vectors such as pSA3 and pAT28.
• Expression vectors are further provided.
• Expression vectors generally are replicable polynucleotide constructs that contain a polynucleotide according to the disclosure.
• Suitable expression vectors include but are not limited to plasmids, viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and expression vector(s) disclosed in PCT Publication No. WO 87/04462.
• Vector components may generally include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcriptional controlling elements (such as promoters, enhancers and terminator).
• suitable transcriptional controlling elements such as promoters, enhancers and terminator
• one or more translational controlling elements are also usually required, such as ribosome binding sites, translation initiation sites, and stop codons.
• the vectors containing the polynucleotides of interest and/or the polynucleotides themselves, can be introduced into the host cell by any of a number of appropriate means, including electroporation, transfection employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; lipofection; and infection (e.g., where the vector is an infectious agent such as vaccinia virus).
• electroporation employing calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances
• microprojectile bombardment e.g., where the vector is an infectious agent such as vaccinia virus.
• infection e.g., where the vector is an infectious agent such as vaccinia virus.
• the choice of introducing vectors or polynucleotides will often depend on features of the host cell.
• the polypeptides and/or fragment thereof may be made recombinantly using a suitable host cell.
• a nucleic acid encoding the polypeptides and/or fragment thereof can be cloned into an expression vector, which can then be introduced into a host cell, such as E. coli cell, a yeast cell, an insect cell, a simian COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell where the cell does not otherwise produce an immunoglobulin protein, to obtain the synthesis of polypeptides and/or fragment thereof in the recombinant host cell.
• a host cell such as E. coli cell, a yeast cell, an insect cell, a simian COS cell, a Chinese hamster ovary (CHO) cell, or a myeloma cell where the cell does not otherwise produce an immunoglobulin protein, to obtain the synthesis of polypeptides and/or fragment thereof in the recombinant host cell
• Preferred host cells include a HT-22 cell, a CHO cell, a Human embryonic kidney HEK-293 cell, or an Sp2.0 cell, among many cells well-known in the art.
• An antibody fragment can be produced by proteolytic or other degradation of a full-length nucleic acid, by recombinant methods, or by chemical synthesis.
• a polypeptide fragment, especially shorter polypeptides up to about 50 amino acids, can be conveniently made by chemical synthesis. Methods of chemical synthesis for proteins and peptides are known in the art and are commercially available.
• polypeptides and/or fragment thereof may be expressed in prokaryotic cells, such as bacterial cells; or in eukaryotic cells, such as fungal cells (such as yeast), plant cells, insect cells, and mammalian cells. Such expression may be earned out, for example, according to procedures known in the art.
• exemplary eukaryotic cells that may be used to express polypeptides include, but are not limited to, COS cells, including COS 7 cells; I 293 cells, including 293-6E cells; CHO cells, including CHO-S, DG44. Lecl3 CHO cells, and FUT8 CHO cells; PER.C6® cells (Crucell); and NSO cells.
• polypeptides and/or fragment thereof may be expressed in yeast. See, e.g, U.S. Publication No. US 2006/0270045 Al.
• a particular eukaryotic host cell is selected based on its ability to make desired post-translational modifications to the anti-CDCPl heavy chains and/or anti-CDCPl light chains.
• CHO cells produce polypeptides that have ahigher level of sialylation than the same polypeptide produced in 293 cells.
• nucleic acids may be transiently or stably transfected in the desired host cells, according to any suitable method.
• Isolated fragments may be purified by any suitable method.
• Many methods of purifying polypeptides are known in the art.
• polypeptides and/or fragment thereof are produced in a cell-free system.
• Non-limiting exemplary cell- free systems are described, e.g., in Sitaraman et al., Methods Mol. Biol. 498: 229-44 (2009); Spirin, Trends Biotechnol. 22: 538-45 (2004); Endo et al., Biotechnol. Adv. 21 : 695-713 (2003).
• the compounds and compositions described herein can be used in methods for treating diseases and/or disorders. In some embodiments, the compounds and compositions described herein can be used in methods for treating diseases associated with PDE11 A4 activity.
• the disclosure relates to a method of treating and/or preventing a disease or disorder alleviated by inhibiting PDE11 A4 activity in a patient in need thereof, including administering to the patient a therapeutically effective amount of a PDE11A4 inhibitor.
• the therapeutically effective amount of the PDE11 A4 inhibitor administered to the patient is capable of changing the subcellular localization and/or location of PDE11A4.
• the therapeutically effective amount of the PDE11 A4 inhibitor administered to the patient is capable of disrupting and/or preventing homodimerization of PDE11 A4.
• the therapeutically effective amount of the PDE11 A4 inhibitor administered to the patient is capable of degrading PDE11 A4.
• the therapeutically effective amount of the PDE11 A4 inhibitor administered to the patient is capable of disrupting and/or preventing homodimerization of PDE11A4, thereby changing the subcellular localization and/or location of PDE11A4 and triggering degradation of PDE11A4.
• the PDE1 1 A4 inhibitor comprises or consists of an isolated fragment of PDE11 A4 comprising a GAF-B binding sequence.
• the PDE11 A4 inhibitor comprises or consists of an isolated fragment of PDE11A4 comprising a GAF-B binding sequence and comprising or consisting of a polypeptide sequence of SEQ ID NO: 1, or a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.
• the PDE11A4 inhibitor comprises or consists of an isolated fragment of PDE11A4 comprising a GAF-B binding sequence and comprising or consisting of a polypeptide sequence of SEQ ID NO: 2, or a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.
• the disease or disorder is associated with cognitive decline.
• the disease or disorder is selected from dementia, Alzheimer’s Disease (AD) including mild Alzheimer's disease and early-onset Alzheimer’s disease, Down’s syndrome, vascular dementia (cerebral amyloid angiopathy and stroke), dementia with Lewy bodies, HIV dementia, Mild Cognitive Impairment (MCI); Age- Associated Memory Impairment (AAMI); Age-Related Cognitive Decline (ARCD) (including age-related cognitive decline of associative long-term memories (aLTMs), dementia associated with traumatic brain injury, preclinical Alzheimer's Disease (PCAD); Cognitive Impairment No Dementia (CIND), and cognitive decline associate with spatial memory, other depression-related behaviors, additional anxiety-related behaviors, sensorimotor gating, and social behaviors.
• AD Alzheimer’s Disease
• AD Alzheimer's Disease
• MCI Mild Cognitive Impairment
• AAMI Age- Associated Memory Impairment
• ARCD Age-Related Cognitive Decline
• ALD Alzheimer-Related Cognitive Decline
• PCAD preclinical Alzheimer's
• cognitive decline includes be any negative change in an animal’s cognitive function.
• cognitive decline includes but is not limited to, memory loss (e.g. behavioral memory loss), failure to acquire new memories, confusion, impaired judgment, personality changes, disorientation, or any combination thereof.
• an active pharmaceutical ingredient or combination of active pharmaceutical ingredients such as any of the PDE11A4 inhibitors of the disclosure (e.g. molecules (e.g. polypeptides) capable of changing the subcellular localization and/or location of PDE11A4, and/or disrupting and/or preventing homodimerization of PDE11A4, and/or degrading PDE1 1 A4), is provided as a pharmaceutically acceptable composition.
• the PDE11A4 inhibitor comprises or consists of an isolated fragment of PDE11A4 comprising a GAF-B binding sequence (e.g. SEQ ID NO: 1 or SEQ ID NO: 2).
• the disclosure relates to a pharmaceutical composition including a therapeutically effective amount of a PDE11 A4 inhibitor for the treatment of a disease alleviated by inhibiting PDE11 A4 activity (e.g. changing the subcellular localization and/or location of PDE11A4, and/or disrupting and/or preventing homodimerization of PDE11 A4, and/or degrading PDE11 A4) in a patient in need thereof, and a physiologically compatible earner medium.
• the disease is associated with cognitive decline.
• the PDE11A4 inhibitor comprises or consists of an isolated fragment of PDE11A4 comprising a GAF-B binding sequence (e.g. SEQ ID NO: 1 or SEQ ID NO: 2).
• the disease or disorder is selected from dementia, Alzheimer’s Disease (AD) including mild Alzheimer's disease and early-onset Alzheimer’s disease, Down’s syndrome, vascular dementia (cerebral amyloid angiopathy and stroke), dementia with Lewy bodies, HIV dementia, Mild Cognitive Impairment (MCI); Age- Associated Memory Impairment (AAMI); Age-Related Cognitive Decline (ARCD) (including age-related cognitive decline of associative long-term memories (aLTMs), dementia associated with traumatic brain injury, prechnical Alzheimer's Disease (PCAD); Cognitive Impairment No Dementia (CIND), and cognitive decline associate with spatial memory, other depression-related behaviors, additional anxiety-related behaviors, sensorimotor gating, and social behaviors.
• AD Alzheimer’s Disease
• AD Alzheimer's Disease
• MCI Mild Cognitive Impairment
• AAMI Age- Associated Memory Impairment
• ARCD Age-Related Cognitive Decline
• ALD Alzheimer-Related Cognitive Decline
• PCAD cognitive Impairment No
• the concentration of each of the active pharmaceutical ingredients provided in the pharmaceutical compositions of the disclosure such as any of the PDE11A4 inhibitors of the disclosure, for example an isolated fragment of PDE11A4 comprising a GAF-B binding sequence (e.g.
• SEQ ID NO: 1 or SEQ ID NO: 2) is less than, for example, 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.09%, 0.08%, 0.07%, 0.06%, 0.05%, 0.04%, 0.03%, 0.02%, 0.01%, 0.009%, 0.008%, 0.007%, 0.006%, 0.005%, 0.004%, 0.003%, 0.002%, 0.001%, 0.0009%, 0.0008%, 0.0007%, 0.0006%, 0.0005%, 0.0004%, 0.0003%, 0.0002% or 0.0001% w/w, w/v, or v/v of the pharmaceutical composition.
• the concentration of each of the active pharmaceutical ingredients provided in the pharmaceutical compositions of the disclosure such as any of the PDE11 A4 inhibitors of the disclosure, for example an isolated fragment of PDE11 A4 comprising a GAF-B binding sequence (e g.
• SEQ ID NO: 1 or SEQ ID NO: 2) is greater than 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 19.75%, 19.50%, 19.25% 19%, 18.75%, 18.50%, 18.25% 18%, 17.75%, 17.50%, 17.25% 17%, 16.75%, 16.50%, 16.25% 16%, 15.75%, 15.50%, 15.25% 15%, 14.75%, 14.50%, 14.25% 14%, 13.75%, 13.50%, 13.25% 13%, 12.75%, 12.50%, 12.25% 12%, 11.75%, 11.50%, 11.25% 11%, 10.75%, 10.50%, 10.25% 10%, 9.75%, 9.50%, 9.25% 9%, 8.75%, 8.50%, 8.25% 8%, 7.75%, 7.50%, 7.25% 7%, 6.75%, 6.50%, 6.25% 6%, 5.75%, 5.50%, 5.25% 5%, 4.75%, 4.50%, 4.25%,
• the concentration of each of the active pharmaceutical ingredients provided in the pharmaceutical compositions of the disclosure such as any of the PDE11A4 inhibitors of the disclosure, for example an isolated fragment of PDE11A4 comprising a GAF-B binding sequence (e.g.
• SEQ ID NO: 1 or SEQ ID NO: 2) is in the range from about 0.0001% to about 50%, about 0.001% to about 40%, about 0.01% to about 30%, about 0.02% to about 29%, about 0.03% to about 28%, about 0.04% to about 27%, about 0.05% to about 26%, about 0.06% to about 25%, about 0.07% to about 24%, about 0.08% to about 23%, about 0.09% to about 22%, about 0.1% to about 21%, about 0.2% to about 20%, about 0.3% to about 19%, about 0.4% to about 18%, about 0.5% to about 17%, about 0.6% to about 16%, about 0.7% to about 15%, about 0.8% to about 14%, about 0.9% to about 12% or about 1% to about 10% w/w, w/v, or v/v of the pharmaceutical composition.
• the concentration of each of the active pharmaceutical ingredients provided in the pharmaceutical compositions of the disclosure such as any of the PDE11A4 inhibitors of the disclosure, for example an isolated fragment of PDE11A4 comprising a GAF-B binding sequence (e.g.
• SEQ ID NO: 1 or SEQ ID NO: 2) is in the range from about 0.001% to about 10%, about 0.01% to about 5%, about 0.02% to about 4.5%, about 0.03% to about 4%, about 0.04% to about 3.5%, about 0.05% to about 3%, about 0.06% to about 2.5%, about 0.07% to about 2%, about 0.08% to about 1.5%, about 0.09% to about 1%, about 0.1% to about 0.9% w/w, w/v, or v/v of the pharmaceutical composition.
• the amount of each of the active pharmaceutical ingredients provided in the pharmaceutical compositions of the disclosure such as any of the foregoing PDE11 A4 inhibitors of the disclosure, for example an isolated fragment of PDE1 1 A4 comprising a GAF-B binding sequence (e g.
• SEQ ID NO: 1 or SEQ ID NO: 2) is equal to or less than 10 g, 9.5 g, 9.0 g, 8.5 g, 8.0 g, 7.5 g, 7.0 g, 6.5 g, 6.0 g, 5.5 g, 5.0 g, 4.5 g, 4.0 g, 3.5 g, 3.0 g, 2.5 g, 2.0 g, 1.5 g, 1.0 g, 0.95 g, 0.9 g, 0.85 g, 0.8 g, 0.75 g, 0.7 g, 0.65 g, 0.6 g, 0.55 g, 0.5 g, 0.45 g, 0.4 g, 0.35 g, 0.3 g, 0.25 g, 0.2 g, 0.15 g, 0.1 g, 0.09 g, 0.08 g, 0.07 g, 0.06 g, 0.05 g, 0.04 g, 0.03 g, 0.02 g, 0.01 g, 0.009 g, 0.008
• the amount of each of the active pharmaceutical ingredients provided in the pharmaceutical compositions of the disclosure such as any of the PDE11A4 inhibitors of the disclosure, for example an isolated fragment of PDE11A4 comprising a GAF-B binding sequence (e.g.
• SEQ ID NO: 1 or SEQ ID NO: 2) is more than 0.0001 g, 0.0002 g, 0.0003 g, 0.0004 g, 0.0005 g, 0.0006 g, 0.0007 g, 0.0008 g, 0.0009 g, 0.001 g, 0.0015 g, 0.002 g, 0.0025 g, 0.003 g, 0.0035 g, 0.004 g, 0.0045 g, 0.005 g, 0.0055 g, 0.006 g, 0.0065 g, 0.007 g, 0.0075 g, 0.008 g, 0.0085 g, 0.009 g, 0.0095 g, 0.01 g, 0.015 g, 0.02 g, 0.025 g, 0.03 g, 0.035 g, 0.04 g, 0.045 g, 0.05 g, 0.055 g, 0.06 g, 0.065 g, 0.07 g, 0.075 g
• Each of the active pharmaceutical ingredients according to the disclosure is effective over a wide dosage range.
• dosages independently range from 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, and from 5 to 40 mg per day are examples of dosages that may be used.
• the exact dosage will depend upon the route of administration, the form in which the compound is administered, the gender and age of the subject to be treated, the body weight of the subject to be treated, and the preference and experience of the attending physician.
• the clinically-established dosages of the PDE11A4 inhibitors of the disclosure may also be used if appropriate.
• the molar ratio of two active pharmaceutical ingredients in the pharmaceutical compositions is in the range from 10: 1 to 1:10, preferably from 2.5: 1 to 1:2.5, and more preferably about 1 : 1.
• the weight ratio of the molar ratio of two active pharmaceutical ingredients in the pharmaceutical compositions is selected from the group consisting of 20: 1, 19:1, 18:1, 17:1, 16:1, 15: 1, 14: 1, 13: 1, 12: 1, 11: 1, 10: 1, 9: 1, 8:1, 7: 1, 6: 1, 5: 1, 4:1, 3: 1, 2: 1, 1 :1, 1:2, 1:3, 1 :4, 1:5, 1:6, 1:7, 1 :8, 1:9, 1: 10, 1: 11, 1: 12, 1: 13, 1: 14, 1 : 15, 1 : 16, 1: 17, 1: 18, 1: 19, and 1 :20.
• the weight ratio of the molar ratio of two active pharmaceutical ingredients in the pharmaceutical compositions is selected from the group consisting of 20: 1, 19: 1, 18: 1, 17: 1, 16: 1, 15: 1, 14: 1, 13: 1, 12:1, 11: 1, 10: 1, 9: 1, 8: 1, 7: 1, 6:1, 5: 1, 4: 1, 3:1, 2: 1, 1: 1, 1 :2, 1:3, 1:4, 1:5, 1 :6, 1:7, 1:8, 1 :9, 1: 10, 1:11, 1:12, 1: 13, 1 : 14, 1 :15, 1:16, 1:17, 1:18, 1: 19, and 1 :20.
• compositions described herein such as any of the PDE11A4 inhibitors of the disclosure, for example an isolated fragment of PDE11A4 comprising a GAF-B binding sequence (e.g. SEQ ID NO: 1 or SEQ ID NO: 2), are for use in the treatment of a disease or disorder associated with cognitive decline.
• a GAF-B binding sequence e.g. SEQ ID NO: 1 or SEQ ID NO: 2
• the pharmaceutical compositions described herein are for use in the treatment of dementia, Alzheimer’s Disease (AD) including mild Alzheimer's disease and early-onset Alzheimer’s disease, Down's syndrome, vascular dementia (cerebral amyloid angiopathy and stroke), dementia with Lewy bodies, HIV dementia, Mild Cognitive Impairment (MCI); Age-Associated Memory Impairment (AAMI); Age-Related Cognitive Decline (ARCD) (including age-related cognitive decline of associative long-term memories (aLTMs), dementia associated with traumatic brain injury, preclinical Alzheimer's Disease (PCAD); Cognitive Impairment No Dementia (CIND), or cognitive decline associate with spatial memory, other depression-related behaviors, additional anxiety-related behaviors, sensorimotor gating, and social behaviors.
• AD Alzheimer’s Disease
• MCI Mild Cognitive Impairment
• AAMI Age-Associated Memory Impairment
• ARCD Age-Related Cognitive Decline
• ALTMs associative long-term memories
• PCAD preclinical Alzheimer's Disease
• compositions for Oral Administration are provided.
• the disclosure provides a pharmaceutical composition for oral administration containing the active pharmaceutical ingredient or combination of active pharmaceutical ingredients, such as the PDE11 A4 inhibitors described herein (e.g. molecules capable of changing the subcellular localization and/or location of PDE11A4, and/or disrupting and/or preventing homodimerization of PDE11A4, and/or degrading PDE11A4), and a pharmaceutical excipient suitable for oral administration.
• active pharmaceutical ingredients such as the PDE11 A4 inhibitors described herein (e.g. molecules capable of changing the subcellular localization and/or location of PDE11A4, and/or disrupting and/or preventing homodimerization of PDE11A4, and/or degrading PDE11A4)
• a pharmaceutical excipient suitable for oral administration e.g. molecules capable of changing the subcellular localization and/or location of PDE11A4, and/or disrupting and/or preventing homodimerization of PDE11A4, and/or degrading PDE11A4
• the disclosure provides a solid pharmaceutical composition for oral administration containing: (i) an effective amount of an active pharmaceutical ingredient or combination of active pharmaceutical ingredients, and (ii) a pharmaceutical excipient suitable for oral administration.
• the composition further contains (hi) an effective amount of a third active pharmaceutical ingredient, and optionally (iv) an effective amount of a fourth active pharmaceutical ingredient.
• the pharmaceutical composition may be a liquid pharmaceutical composition suitable for oral consumption.
• Pharmaceutical compositions of the disclosure suitable for oral administration can be presented as discrete dosage forms, such as capsules, sachets, or tablets, or liquids or aerosol sprays each containing a predetermined amount of an active ingredient as a powder or in granules, a solution, or a suspension in an aqueous or non-aqueous liquid, an oil-in-water emulsion, a water-in-oil liquid emulsion, powders for reconstitution, powders for oral consumptions, bottles (including powders or liquids in a bottle), orally dissolving films, lozenges, pastes, tubes, gums, and packs.
• Such dosage forms can be prepared by any of the methods of pharmacy, but all methods include the step of bringing the active ingredient(s) into association with the carrier, which constitutes one or more necessary ingredients.
• the compositions are prepared by uniformly and intimately admixing the active ingredient(s) with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product into the desired presentation.
• a tablet can be prepared by compression or molding, optionally with one or more accessory ingredients.
• Compressed tablets can be prepared by compressing in a suitable machine the active ingredient in a free-flowing form such as powder or granules, optionally mixed with an excipient such as, but not limited to, a binder, a lubricant, an inert diluent, and/or a surface active or dispersing agent. Molded tablets can be made by molding in a suitable machine a mixture of the powdered compound moistened with an inert liquid diluent. [00118] The disclosure further encompasses anhydrous pharmaceutical compositions and dosage forms since water can facilitate the degradation of some compounds.
• water may be added (e.g, 5%) in the pharmaceutical arts as a means of simulating long-term storage in order to determine characteristics such as shelf-life or the stability of formulations over time.
• Anhydrous pharmaceutical compositions and dosage forms of the disclosure can be prepared using anhydrous or low moisture containing ingredients and low moisture or low humidity conditions.
• Pharmaceutical compositions and dosage forms of the disclosure which contain lactose can be made anhydrous if substantial contact with moisture and/or humidity during manufacturing, packaging, and/or storage is expected.
• An anhydrous pharmaceutical composition may be prepared and stored such that its anhydrous nature is maintained. Accordingly, anhydrous compositions may be packaged using materials known to prevent exposure to water such that they can be included in suitable formulary kits. Examples of suitable packaging include, but are not limited to, hermetically sealed foils, plastic or the like, unit dose containers, blister packs, and strip packs.
• Each of the active pharmaceutical ingredients can be combined in an intimate admixture with a pharmaceutical carrier according to conventional pharmaceutical compounding techniques.
• the carrier can take a wide variety of forms depending on the form of preparation desired for administration.
• any of the usual pharmaceutical media can be employed as carriers, such as, for example, water, glycols, oils, alcohols, flavoring agents, preservatives, coloring agents, and the like in the case of oral liquid preparations (such as suspensions, solutions, and elixirs) or aerosols; or carriers such as starches, sugars, micro-crystalline cellulose, diluents, granulating agents, lubricants, binders, and disintegrating agents can be used in the case of oral solid preparations, in some embodiments without employing the use of lactose
• suitable carriers include powders, capsules, and tablets, with the solid oral preparations. If desired, tablets can be coated by standard aqueous or nonaqueous techniques.
• Binders suitable for use in pharmaceutical compositions and dosage forms include, but are not limited to, com starch, potato starch, or other starches, gelatin, natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyvinyl pyrrolidone, methyl cellulose, pre-gelatinized starch, hydroxypropyl methyl cellulose, microcrystallme cellulose, and mixtures thereof.
• natural and synthetic gums such as acacia, sodium alginate, alginic acid, other alginates, powdered tragacanth, guar gum, cellulose and its derivatives (e.g., ethyl cellulose, cellulose acetate, carboxymethyl cellulose calcium, sodium carboxymethyl cellulose), polyviny
• suitable fillers for use in the pharmaceutical compositions and dosage forms disclosed herein include, but are not limited to, talc, calcium carbonate (e.g, granules or powder), microcrystalline cellulose, powdered cellulose, dextrates, kaolin, mannitol, silicic acid, sorbitol, starch, pre-gelatinized starch, and mixtures thereof.
• Disintegrants may be used in the compositions of the disclosure to provide tablets that disintegrate when exposed to an aqueous environment. Too much of a disintegrant may produce tablets which disintegrate in the bottle. Too little may be insufficient for disintegration to occur, thus altering the rate and extent of release of the active ingredients from the dosage form. Thus, a sufficient amount of disintegrant that is neither too little nor too much to detrimentally alter the release of the active ingredient(s) may be used to form the dosage forms of the compounds disclosed herein. The amount of disintegrant used may vary based upon the type of formulation and mode of administration, and may be readily discernible to those of ordinary skill in the art.
• Disintegrants that can be used to form pharmaceutical compositions and dosage forms of the disclosure include, but are not limited to, agar-agar, alginic acid, calcium carbonate, microcrystalline cellulose, croscarmellose sodium, crospovidone, polacrilin potassium, sodium starch glycolate, potato or tapioca starch, other starches, pre-gelatinized starch, other starches, clays, other algins, other celluloses, gums or mixtures thereof.
• Lubricants which can be used to form pharmaceutical compositions and dosage forms of the disclosure include, but are not limited to, calcium stearate, magnesium stearate, sodium stearyl fumarate, mineral oil, light mineral oil, glycerin, sorbitol, mannitol, polyethylene glycol, other glycols, stearic acid, sodium lauryl sulfate, talc, hydrogenated vegetable oil (e g., peanut oil, cottonseed oil, sunflower oil, sesame oil, olive oil, com oil, and soybean oil), zinc stearate, ethyl oleate, ethylaureate, agar, or mixtures thereof.
• Additional lubricants include, for example, a syloid silica gel, a coagulated aerosol of synthetic silica, silicified microcrystalline cellulose, or mixtures thereof.
• a lubricant can optionally be added in an amount of less than about 0.5% or less than about 1% (by weight) of the pharmaceutical composition.
• the active pharmaceutical ingredient(s) may be combined with various sweetening or flavoring agents, coloring matter or dyes and, if so desired, emulsifying and/or suspending agents, together with such diluents as water, ethanol, propylene glycol, glycerin and various combinations thereof.
• the tablets can be uncoated or coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period.
• a time delay material such as glyceryl monostearate or glyceryl distearate can be employed.
• Formulations for oral use can also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with water or an oil medium, for example, peanut oil, liquid paraffin or olive oil.
• Surfactants which can be used to form pharmaceutical compositions and dosage forms of the disclosure include, but are not limited to, hydrophilic surfactants, lipophilic surfactants, and mixtures thereof. That is, a mixture of hydrophilic surfactants may be employed, a mixture of lipophilic surfactants may be employed, or a mixture of at least one hydrophilic surfactant and at least one lipophilic surfactant may be employed.
• a suitable hydrophilic surfactant may generally have an HLB value of at least 10, while suitable lipophilic surfactants may generally have an HLB value of or less than about 10.
• An empirical parameter used to characterize the relative hydrophilicity and hydrophobicity of non-ionic amphiphilic compounds is the hydrophilic-lipophilic balance (“HLB” value).
• HLB hydrophilic-lipophilic balance
• Surfactants with lower HLB values are more lipophilic or hydrophobic, and have greater solubility in oils, while surfactants with higher HLB values are more hydrophilic, and have greater solubility in aqueous solutions.
• Hydrophilic surfactants are generally considered to be those compounds having an HLB value greater than about 10, as well as anionic, cationic, or zwitterionic compounds for which the HLB scale is not generally applicable.
• lipophilic (z.e., hydrophobic) surfactants are compounds having an HLB value equal to or less than about 10.
• HLB value of a surfactant is merely a rough guide generally used to enable formulation of industrial, pharmaceutical and cosmetic emulsions.
• Hydrophilic surfactants may be either ionic or non-ionic. Suitable ionic surfactants include, but are not limited to, alkylammonium salts; fusidic acid salts; fatty acid derivatives of amino acids, oligopeptides, and polypeptides; glyceride derivatives of amino acids, oligopeptides, and polypeptides; lecithins and hydrogenated lecithins; lysolecithins and hydrogenated lysolecithins; phospholipids and denvatives thereof; lysophosphohpids and derivatives thereof; carnitine fatty acid ester salts; salts of alkylsulfates; fatty acid salts; sodium docusate; acyl-lactylates; mono- and di-acetylated tartaric acid esters of mono- and di-glycerides; succinylated mono- and di-glycerides; citric acid esters of mono- and digly
• ionic surfactants include, by way of example: lecithins, lysolecithin, phospholipids, lysophosphohpids and derivatives thereof; carnitine fatty acid ester salts; salts of alkylsulfates; fatty acid salts; sodium docusate; acylactylates; mono- and di-acetylated tartaric acid esters of mono- and di-glycerides; succinylated mono- and di-glycerides; citric acid esters of mono- and di-glycerides; and mixtures thereof.
• Ionic surfactants may be the ionized forms of lecithin, lysolecithin, phosphatidylcholine, phosphatidylethanolamine, phosphatidylglycerol, phosphatidic acid, phosphatidylserine, lysophosphatidyl choline, lysophosphatidylethanolamine, lysophosphatidylglycerol, lysophosphatidic acid, lysophosphatidylserine, PEG- phosphatidylethanolamine, PVP-phosphatidylethanolamine, lactylic esters of fatty acids, stearoyl-2-lactylate, stearoyl lactylate, succinylated monoglycerides, mono/diacetylated tartaric acid esters of mono/diglycerides, citric acid esters of mono/diglycerides, cholylsarcosine, caproate, capry
• Hydrophilic non-ionic surfactants may include, but not limited to, alkylglucosides; alkylmaltosides; alkylthioglucosides; lauryl macrogolglycerides; polyoxyalkylene alkyl ethers such as polyethylene glycol alkyl ethers; polyoxyalkylene alkylphenols such as polyethylene glycol alkyl phenols; poly oxyalkylene alkyl phenol fatty acid esters such as polyethylene glycol fatty acids monoesters and polyethylene glycol fatty acids diesters; polyethylene glycol glycerol fatty acid esters; poly glycerol fatty acid esters; polyoxyalkylene sorbitan fatty acid esters such as polyethylene glycol sorbitan fatty acid esters; hydrophilic transesterification products of a polyol with at least one member of the group consisting of glycerides, vegetable oils, hydrogenated vegetable oils, fatty acids, and sterols; polyoxyethylene glycol sorb
• hydrophilic-non-ionic surfactants include, without limitation, PEG-10 laurate, PEG-12 laurate, PEG-20 laurate, PEG-32 laurate, PEG-32 dilaurate, PEG-12 oleate, PEG-15 oleate, PEG-20 oleate, PEG-20 dioleate, PEG-32 oleate, PEG-200 oleate, PEG-400 oleate, PEG- 15 stearate, PEG-32 distearate, PEG-40 stearate, PEG- 100 stearate, PEG-20 dilaurate, PEG-25 glyceryl trioleate, PEG-32 dioleate, PEG-20 glyceryl laurate, PEG-30 glyceryl laurate, PEG-20 glycery l stearate, PEG-20 glyceryl oleate, PEG-30 glyceryl oleate, PEG-30 glyceryl
• Suitable lipophilic surfactants include, by way of example only: fatty alcohols; glycerol fatty acid esters; acetylated glycerol fatty acid esters; lower alcohol fatty acids esters; propylene glycol fatty acid esters; sorbitan fatty acid esters; polyethylene glycol sorbitan fatty acid esters; sterols and sterol derivatives; polyoxyethylated sterols and sterol derivatives; polyethylene glycol alkyl ethers; sugar esters; sugar ethers; lactic acid derivatives of mono- and di-glycerides; hydrophobic transesterification products of a polyol with at least one member of the group consisting of glycerides, vegetable oils, hydrogenated vegetable oils, fatty acids and sterols; oil-soluble vitamins/vitamin derivatives; and mixtures thereof.
• preferred lipophilic surfactants include glycerol fatty acid esters, propylene glycol fatty acid esters, and mixtures thereof, or are hydrophobic transesterification products of a polyol with at least one member of the group consisting of vegetable oils, hydrogenated vegetable oils, and triglycerides.
• the composition may include a solubilizer to ensure good solubilization and/or dissolution of the compound of the present disclosure and to minimize precipitation of the compound of the present disclosure. This can be especially important for compositions for non-oral use - e.g , compositions for injection.
• a solubilizer may also be added to increase the solubility of the hydrophilic drug and/or other components, such as surfactants, or to maintain the composition as a stable or homogeneous solution or dispersion.
• solubilizers include, but are not limited to, the following: alcohols and polyols, such as ethanol, isopropanol, butanol, benzyl alcohol, ethylene glycol, propylene glycol, butanediols and isomers thereof, glycerol, pentaerythritol, sorbitol, mannitol, transcutol, dimethyl isosorbide, polyethylene glycol, polypropylene glycol, polyvinylalcohol, hydroxypropyl methyl cellulose and other cellulose derivatives, cyclodextrins and cyclodextrin derivatives; ethers of polyethylene glycols having an average molecular weight of about 200 to about 6000, such as tetrahydrofurfuryl alcohol PEG ether (gly cofurol) or methoxy PEG; amides and other nitrogen-containing compounds such as 2- pyrrolidone, 2-piperidone, E
• N-alkylpyrrolidone N-hydroxyalkylpyrrolidone, N-alkylpiperidone, N-alkyl caprolactam, dimethylacetamide and polyvinylpyrrolidone
• esters such as ethyl propionate, tributylcitrate, acetyl triethylcitrate, acetyl tributyl citrate, tri ethyl citrate, ethyl oleate, ethyl caprylate, ethyl butyrate, triacetin, propylene glycol monoacetate, propylene glycol diacetate, e-caprolactone and isomers thereof, 5-valerolactone and isomers thereof, P-butyrolactone and isomers thereof; and other solubilizers known in the art, such as dimethyl acetamide, dimethyl isosorbide, N-methyl pyrrolidones, monooctanoin, diethylene
• solubilizers may also be used. Examples include, but not limited to, triacetin, triethylcitrate, ethyl oleate, ethyl caprylate, dimethylacetamide, N- methylpyrrolidone, N-hydroxyethylpyrrolidone, polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropyl cyclodextrins, ethanol, polyethylene glycol 200-100, glycofurol, transcutol, propylene glycol, and dimethyl isosorbide. Particularly preferred solubilizers include sorbitol, glycerol, triacetin, ethyl alcohol, PEG-400, glycofurol and propylene glycol.
• the amount of solubilizer that can be included is not particularly limited.
• the amount of a given solubilizer may be limited to a bioacceptable amount, which may be readily determined by one of skill in the art.
• the solubilizer can be in a weight ratio of 10%, 25%, 50%, 100%, or up to about 200% by weight, based on the combined weight of the drug, and other excipients.
• very small amounts of solubilizer may also be used, such as 5%, 2%, 1% or even less.
• the solubilizer may be present in an amount of about 1% to about 100%, more typically about 5% to about 25% by weight.
• the composition can further include one or more pharmaceutically acceptable additives and excipients.
• additives and excipients include, without limitation, detackifiers, anti-foaming agents, buffering agents, polymers, antioxidants, preservatives, chelating agents, viscomodulators, tonicifiers, flavorants, colorants, odorants, opacifiers, suspending agents, binders, fillers, plasticizers, lubricants, and mixtures thereof.
• an acid or a base may be incorporated into the composition to facilitate processing, to enhance stability , or for other reasons.
• pharmaceutically acceptable bases include amino acids, amino acid esters, ammonium hydroxide, potassium hydroxide, sodium hydroxide, sodium hydrogen carbonate, aluminum hydroxide, calcium carbonate, magnesium hydroxide, magnesium aluminum silicate, synthetic aluminum silicate, synthetic hydrocalcite, magnesium aluminum hydroxide, diisopropylethylamine, ethanolamine, ethylenediamine, triethanolamine, triethylamine, triisopropanolamine, trimethylamine, tris(hydroxymethyl)aminomethane (TRIS) and the like.
• bases that are salts of a pharmaceutically acceptable acid, such as acetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonic acid, amino acids, ascorbic acid, benzoic acid, boric acid, butyric acid, carbonic acid, citric acid, fatty acids, formic acid, fumaric acid, gluconic acid, hydroquinosulfonic acid, isoascorbic acid, lactic acid, maleic acid, oxalic acid, parabromophenylsulfonic acid, propionic acid, p-toluenesulfonic acid, salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid, thiogly colic acid, toluenesulfonic acid, uric acid, and the like.
• a pharmaceutically acceptable acid such as acetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonic acid, amino acids,
• Salts of polyprotic acids such as sodium phosphate, disodium hydrogen phosphate, and sodium dihydrogen phosphate can also be used.
• the cation can be any convenient and pharmaceutically acceptable cation, such as ammonium, alkali metals and alkaline earth metals.
• Example may include, but not limited to, sodium, potassium, lithium, magnesium, calcium and ammonium.
• Suitable acids are pharmaceutically acceptable organic or inorganic acids.
• suitable inorganic acids include hydrochloric acid, hydrobromic acid, hydriodic acid, sulfuric acid, nitric acid, boric acid, phosphoric acid, and the like.
• suitable organic acids include acetic acid, acrylic acid, adipic acid, alginic acid, alkanesulfonic acids, amino acids, ascorbic acid, benzoic acid, boric acid, butyric acid, carbonic acid, citric acid, fatty acids, formic acid, fumaric acid, gluconic acid, hydroquinosulfonic acid, isoascorbic acid, lactic acid, maleic acid, methanesulfonic acid, oxalic acid, para-bromophenylsulfonic acid, propionic acid, p-toluenesulfonic acid, salicylic acid, stearic acid, succinic acid, tannic acid, tartaric acid, thiogly colic acid, toluenesulfonic acid and uric acid.
• a pharmaceutical composition for injection containing an active pharmaceutical ingredient or combination of active pharmaceutical ingredients, such as a PDE11A4 inhibitors of the disclosure (e.g., molecules capable of changing the subcellular localization and/or location of PDE11A4, and/or disrupting and/or preventing homodimerization of PDE11 A4, and/or degrading PDE11A4), for example an isolated fragment of PDE11A4 comprising a GAF-B binding sequence (e.g. SEQ ID NO: 1 or SEQ ID NO: 2), and a pharmaceutical excipient suitable for injection.
• a PDE11A4 inhibitors of the disclosure e.g., molecules capable of changing the subcellular localization and/or location of PDE11A4, and/or disrupting and/or preventing homodimerization of PDE11 A4, and/or degrading PDE11A4
• an isolated fragment of PDE11A4 comprising a GAF-B binding sequence (e.g. SEQ ID NO: 1 or SEQ ID NO: 2), and
• Aqueous solutions in saline are also conventionally used for injection.
• Ethanol, glycerol, propylene glycol and liquid polyethylene glycol (and suitable mixtures thereof), cyclodextrin derivatives, and vegetable oils may also be employed.
• the proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, for the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, and thimerosal.
• Sterile injectable solutions are prepared by incorporating an active pharmaceutical ingredient or combination of active pharmaceutical ingredients in the required amounts in the appropriate solvent with various other ingredients as enumerated above, as required, followed by filtered sterilization.
• dispersions are prepared by incorporating the various stenhzed active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
• certain desirable methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
• a pharmaceutical composition for transdermal delivery containing an active pharmaceutical ingredient or combination of active pharmaceutical ingredients, such as PDE11A4 inhibitors of the disclosure (e.g. molecules capable of changing the subcellular localization and/or location of PDE11A4, and/or disrupting and/or preventing homodimerization of PDE11A4, and/or degrading PDE11A4), for example an isolated fragment of PDE11A4 comprising a GAF-B binding sequence (e.g. SEQ ID NO: 1 or SEQ ID NO: 2), and a pharmaceutical excipient suitable for transdermal delivery.
• PDE11A4 inhibitors of the disclosure e.g. molecules capable of changing the subcellular localization and/or location of PDE11A4, and/or disrupting and/or preventing homodimerization of PDE11A4, and/or degrading PDE11A4
• an isolated fragment of PDE11A4 comprising a GAF-B binding sequence (e.g. SEQ ID NO: 1 or SEQ ID NO: 2), and
• compositions of the present disclosure can be formulated into preparations in solid, semi-solid, or liquid forms suitable for local or topical administration, such as gels, water soluble jellies, creams, lotions, suspensions, foams, powders, slurries, ointments, solutions, oils, pastes, suppositories, sprays, emulsions, saline solutions, dimethylsulfoxide (DMSO)-based solutions.
• DMSO dimethylsulfoxide
• carriers with higher densities are capable of providing an area with a prolonged exposure to the active ingredients.
• a solution formulation may provide more immediate exposure of the active ingredient to the chosen area.
• compositions also may comprise suitable solid or gel phase carriers or excipients, which are compounds that allow increased penetration of, or assist in the delivery of, therapeutic molecules across the stratum comeum permeability barrier of the skin.
• suitable solid or gel phase carriers or excipients which are compounds that allow increased penetration of, or assist in the delivery of, therapeutic molecules across the stratum comeum permeability barrier of the skin.
• penetration-enhancing molecules known to those trained in the art of topical formulation.
• humectants e.g., urea
• glycols e.g, propylene glycol
• alcohols e.g, ethanol
• fatty acids e.g., oleic acid
• surfactants e.g., isopropyl myristate and sodium lauryl sulfate
• pyrrolidones e.g., isopropyl myristate and sodium lauryl sulfate
• pyrrolidones e.glycerol monolaurate, sulfoxides, terpenes (e.g. , menthol)
• amines amides, alkanes, alkanols, water, calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols.
• transdermal delivery devices Such transdermal patches may be used to provide continuous or discontinuous infusion of an active pharmaceutical ingredient or combination of active pharmaceutical ingredients in controlled amounts, either with or without another active pharmaceutical ingredient.
• transdermal patches for the delivery' of pharmaceutical agents is well known in the art. See, e.g., U.S. Patent Nos. 5,023,252; 4,992,445; and 5,001,139, the entirety of which are incorporated herein by reference. Such patches may be constructed for continuous, pulsatile, or on demand delivery of pharmaceutical agents.
• compositions for Inhalation are provided.
• compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders.
• the liquid or solid compositions may contain suitable pharmaceutically acceptable excipients as described supra and PDE11 A4 inhibitors of the disclosure (e.g. molecules capable of changing the subcellular localization and/or location of PDE11A4, and/or disrupting and/or preventing homodimerization of PDE11 A4, and/or degrading PDE11A4), for example an isolated fragment of PDE11A4 comprising a GAF-B binding sequence (e.g. SEQ ID NO: 1 or SEQ ID NO: 2).
• compositions are administered by the oral or nasal respiratory route for local or systemic effect.
• Compositions in preferably pharmaceutically acceptable solvents may be nebulized by use of inert gases. Nebulized solutions may be inhaled directly from the nebulizing device or the nebulizing device may be attached to a face mask tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from devices that deliver the formulation in an appropriate manner. Dry powder inhalers may also be used to provide inhaled delivery of the compositions.
• compositions of the PDE11 A4 inhibitors of the disclosure e.g. molecules capable of changing the subcellular localization and/or location of PDE1 1 A4, and/or disrupting and/or preventing homodimerization of PDE11A4, and/or degrading PDE11 A4
• an isolated fragment of PDE11A4 comprising a GAF-B binding sequence e.g. SEQ ID NO: 1 or SEQ ID NO: 2
• compositions described herein may also be prepared from compositions described herein and one or more pharmaceutically acceptable excipients suitable for sublingual, buccal, rectal, intraosseous, intraocular, intranasal, epidural, or intraspinal administration. Preparations for such pharmaceutical compositions are well-known in the art.
• Administration of an active pharmaceutical ingredient or combination of active pharmaceutical ingredients or a pharmaceutical composition thereof can be effected by any method that enables delivery of the compounds to the site of action. These methods include oral routes, intraduodenal routes, parenteral injection (including intravenous, intraarterial, subcutaneous, intramuscular, intravascular, intraperitoneal or infusion), topical (e.g., transdermal application), rectal administration, via local delivery by catheter or stent or through inhalation.
• the active pharmaceutical ingredient or combination of active pharmaceutical ingredients can also be administered intraadiposally or intrathecally.
• Exemplary parenteral administration forms include solutions or suspensions of active compound in sterile aqueous solutions, for example, aqueous propylene glycol or dextrose solutions. Such dosage forms can be suitably buffered, if desired. Kits
• kits include an active pharmaceutical ingredient or combination of active pharmaceutical ingredients, either alone or in combination in suitable packaging, and written material that can include instructions for use, discussion of clinical studies and listing of side effects.
• kits may also include information, such as scientific literature references, package insert materials, clinical trial results, and/or summaries of these and the like, which indicate or establish the activities and/or advantages of the composition, and/or which describe dosing, administration, side effects, drug interactions, or other information useful to the health care provider. Such information may be based on the results of various studies, for example, studies using experimental animals involving in vivo models and studies based on human clinical trials.
• the kit may further contain another active pharmaceutical ingredient.
• an active pharmaceutical ingredient or combination of active pharmaceutical ingredients are provided as separate compositions in separate containers within the kit. In selected embodiments, an active pharmaceutical ingredient or combination of active pharmaceutical ingredients are provided as a single composition within a container in the kit.
• Suitable packaging and additional articles for use e.g, measuring cup for liquid preparations, foil wrapping to minimize exposure to air, and the like
• Kits described herein can be provided, marketed and/or promoted to health providers, including physicians, nurses, pharmacists, formulary officials, and the like. Kits may also, in selected embodiments, be marketed directly to the consumer.
• the disclosure provides a kit comprising a composition comprising a therapeutically effective amount of an active pharmaceutical ingredient (e.g, a PDE11 A4 inhibitor of the disclosure, e.g. molecules capable of changing the subcellular localization and/or location of PDE11A4, and/or disrupting and/or preventing homodimerization of PDE11A4, and/or degrading PDE11A4, for example an isolated fragment of PDE11A4 comprising a GAF-B binding sequence, such as SEQ ID NO: 1 or SEQ ID NO: 2), or combination of active pharmaceutical ingredients or a pharmaceutically acceptable salt, solvate, hydrate, cocrystal, or prodrug thereof.
• active pharmaceutical ingredient e.g, a PDE11 A4 inhibitor of the disclosure, e.g. molecules capable of changing the subcellular localization and/or location of PDE11A4, and/or disrupting and/or preventing homodimerization of PDE11A4, and/or degrading PDE11A4, for example an isolated fragment of PDE11
• the disclosure provides a kit comprising (1) a composition comprising a therapeutically effective amount of an active pharmaceutical ingredient (e.g., a PDE11A4 inhibitor of the disclosure, for example an isolated fragment of PDE11A4 comprising a GAF-B binding sequence (e.g. SEQ ID NO: 1 or SEQ ID NO: 2), or combination of active pharmaceutical ingredients, and (2) a diagnostic test for determining whether a patient’s disease or disorder associated with cognitive decline is a particular subtype of a disease or disorder associated with cognitive decline. Any of the foregoing diagnostic methods may be utilized in the kit.
• an active pharmaceutical ingredient e.g., a PDE11A4 inhibitor of the disclosure, for example an isolated fragment of PDE11A4 comprising a GAF-B binding sequence (e.g. SEQ ID NO: 1 or SEQ ID NO: 2), or combination of active pharmaceutical ingredients
• a diagnostic test for determining whether a patient’s disease or disorder associated with cognitive decline is a particular subtype of a disease or disorder associated with cognitive decline. Any
• kits described above are preferably for use in the treatment of the diseases and conditions described herein.
• the kits are for use in the treatment of a disease or disorder associated with cognitive decline .
• the kits are for use in the treatment of dementia, Alzheimer’s Disease (AD) including mild Alzheimer's disease and early-onset Alzheimer’s disease, Down’s syndrome, vascular dementia (cerebral amyloid angiopathy and stroke), dementia with Lewy bodies, HIV dementia, Mild Cognitive Impairment (MCI); Age- Associated Memory Impairment (AAMI); Age-Related Cognitive Decline (ARCD) (including age-related cognitive decline of associative long-term memories (aLTMs), dementia associated with traumatic brain injury, preclinical Alzheimer's Disease (PCAD); Cognitive Impairment No Dementia (C1ND), or cognitive decline associate with spatial memory , other depression-related behaviors, additional anxiety-related behaviors, sensorimotor gating, and social behaviors.
• AD Alzheimer’s Disease
• MCI Mild Cognitive Impairment
• AAMI Age-
• the amounts of the pharmaceutical compositions administered using the methods herein such as the dosages of PDE11A4 inhibitors of the disclosure (e.g., molecules capable of changing the subcellular localization and/or location of PDE11A4, and/or disrupting and/or preventing homodimerization of PDE11A4, and/or degrading PDE11A4), for example an isolated fragment of PDE11 A4 comprising a GAF-B binding sequence (e.g. SEQ ID NO: 1 or SEQ ID NO: 2), will be dependent on the human or mammal being treated, the severity of the disorder or condition, the rate of administration, the disposition of the active pharmaceutical ingredients and the discretion of the prescribing physician.
• PDE11A4 inhibitors of the disclosure e.g., molecules capable of changing the subcellular localization and/or location of PDE11A4, and/or disrupting and/or preventing homodimerization of PDE11A4, and/or degrading PDE11A4
• an effective dosage is in the range of about 0.001 to about 100 mg per kg body weight per day, such as about 1 to about 35 mg/kg/day, in single or divided doses. For a 70 kg human, this would amount to about 0.05 to 7 g/day, such as about 0.05 to about 2.5 g/day. In some instances, dosage levels below the lower limit of the aforesaid range may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect - e.g., by dividing such larger doses into several small doses for administration throughout the day.
• the dosage of the pharmaceutical compositions and active pharmaceutical ingredients may be provided in units of mg/kg of body mass or in mg/m 2 of body surface area.
• the disclosure includes methods of treating a disease or disorder associated with cognitive decline in human subject suffering from the disease or disorder, the method comprising the steps of administering a therapeutically effective dose of an active pharmaceutical ingredient that is a PDE11 A4 inhibitor of the disclosure, for example an isolated fragment of PDE11 A4 comprising a GAF-B binding sequence (e g. SEQ ID NO: 1 or SEQ ID NO: 2), to the human subject.
• a therapeutically effective dose of an active pharmaceutical ingredient that is a PDE11 A4 inhibitor of the disclosure for example an isolated fragment of PDE11 A4 comprising a GAF-B binding sequence (e g. SEQ ID NO: 1 or SEQ ID NO: 2), to the human subject.
• the disclosure includes methods of treating a disease or disorder associated with cognitive decline in a human subject suffering from the disease or disorder, the method comprising the steps of administering a therapeutically effective dose of an active pharmaceutical ingredient that is a PDE11 A4 inhibitor, for example an isolated fragment of PDE11A4 comprising a GAF-B binding sequence (e.g. SEQ ID NO: 1 or SEQ ID NO: 2), to the human subject to inhibit or decrease the activity of PDE11A protein.
• a PDE11 A4 inhibitor for example an isolated fragment of PDE11A4 comprising a GAF-B binding sequence (e.g. SEQ ID NO: 1 or SEQ ID NO: 2)
• a pharmaceutical composition or active pharmaceutical ingredient is administered in a single dose.
• Such administration may be by injection, e.g., intravenous injection, in order to introduce the active pharmaceutical ingredient quickly.
• routes including the preferred oral route, may be used as appropriate.
• a single dose of a pharmaceutical composition may also be used for treatment of an acute condition.
• a pharmaceutical composition or active pharmaceutical ingredient is administered in multiple doses.
• a pharmaceutical composition is administered in multiple doses. Dosing may be once, twice, three times, four times, five times, six times, or more than six times per day. Dosing may be once a month, once every two weeks, once a week, or once every other day. In other embodiments, a pharmaceutical composition is administered about once per day to about 6 times per day. In some embodiments, a pharmaceutical composition is administered once daily, while in other embodiments, a pharmaceutical composition is administered twice daily, and in other embodiments a pharmaceutical composition is administered three times daily.
• a pharmaceutical composition is administered for more than 1, 2, 3, 4, 5, 6, 7, 14, or 28 day(s). In some embodiments, a pharmaceutical composition is administered for less than 28, 14, 7, 6, 5, 4, 3, 2, or 1 day(s). In some embodiments, a pharmaceutical composition is administered chronically on an ongoing basis - e.g., for the treatment of chronic effects. In some embodiments, the administration of a pharmaceutical composition continues for less than about 7 days. In yet another embodiment the administration continues for more than about 6, 10, 14, 28 days, two months, six months, or one year. In some cases, continuous dosing is achieved and maintained as long as necessary.
• an effective dosage of an active pharmaceutical ingredient disclosed herein for example any of the PDE11 A4 inhibitors of the disclosure, for example an isolated fragment of PDE1 1 A4 comprising a GAF-B binding sequence (e.g.
• SEQ ID NO: 1 or SEQ ID NO: 2) is in the range of about 1 mg to about 500 mg, about 10 mg to about 300 mg, about 20 mg to about 250 mg, about 25 mg to about 200 mg, about 10 mg to about 200 mg, about 20 mg to about 150 mg, about 30 mg to about 120 mg, about 10 mg to about 90 mg, about 20 mg to about 80 mg, about 30 mg to about 70 mg, about 40 mg to about 60 mg, about 45 mg to about 55 mg, about 48 mg to about 52 mg, about 50 mg to about 150 mg, about 60 mg to about 140 mg, about 70 mg to about 130 mg, about 80 mg to about 120 mg, about 90 mg to about 110 mg, about 95 mg to about 105 mg, about 150 mg to about 250 mg, about 160 mg to about 240 mg, about 170 mg to about 230 mg, about 180 mg to about 220 mg, about 190 mg to about 210 mg, about 195 mg to about 205 mg, or about 198 to about 202 mg.
• an effective dosage of an active pharmaceutical ingredient disclosed herein is less than about 25 mg, less than about 50 mg, less than about 75 mg, less than about 100 mg, less than about 125 mg, less than about 150 mg, less than about 175 mg, less than about 200 mg, less than about 225 mg, or less than about 250 mg. In some embodiments, an effective dosage of an active pharmaceutical ingredient disclosed herein is greater than about 25 mg, greater than about 50 mg, greater than about 75 mg, greater than about 100 mg, greater than about 125 mg, greater than about 150 mg, greater than about 175 mg, greater than about 200 mg, greater than about 225 mg, or greater than about 250 mg.
• an effective dosage of an active pharmaceutical ingredient disclosed herein for example any of the PDE11 A4 inhibitors of the disclosure, for example an isolated fragment of PDE11 A4 comprising a GAF-B binding sequence (e.g. SEQ ID NO: 1 or SEQ ID NO: 2), is in the range of about 0.01 mg/kg to about 200 mg/kg, or about 0.1 to 100 mg/kg, or about 1 to 50 mg/kg.
• a GAF-B binding sequence e.g. SEQ ID NO: 1 or SEQ ID NO: 2
• an active pharmaceutical ingredient is administered at a dosage of 10 to 200 mg BID, including 50, 60, 70, 80, 90, 100, 150, or 200 mg BID.
• an active pharmaceutical ingredient is administered at a dosage of 10 to 500 mg BID, including 1, 5, 10, 15, 25, 50, 75, 100, 150, 200, 300, 400, or 500 mg BID.
• dosage levels below the lower limit of the aforesaid ranges may be more than adequate, while in other cases still larger doses may be employed without causing any harmful side effect, e.g, by dividing such larger doses into several small doses for administration throughout the day.
• the dosage actually administered will depend upon the condition being treated, the age, health and weight of the recipient, the type of concurrent treatment, if any, and the frequency of treatment.
• the effective dosage amount may be determined by one skilled in the art on the basis of routine empirical activity testing to measure the bioactivity of the compound(s) in a bioassay, and thus establish the appropriate dosage to be administered.
• An effective amount of the combination of the active pharmaceutical ingredient may be administered in either single or multiple doses by any of the accepted modes of administration of agents having similar utilities, including rectal, buccal, intranasal and transdermal routes, by intra-arterial injection, intravenously, intraperitoneally, parenterally, intramuscularly, subcutaneously, orally, topically, or as an inhalant.
• compositions described herein further include controlled-release, sustained release, or extended-release therapeutic dosage forms for administration of the compounds described herein, which involves incorporation of the compounds into a suitable delivery system in the formation of certain compositions.
• This dosage form controls release of the compound(s) in such a manner that an effective concentration of the compound(s) in the bloodstream may be maintained over an extended period of time, with the concentration in the blood remaining relatively constant, to improve therapeutic results and/or minimize side effects.
• a controlled-release system would provide minimum peak to trough fluctuations in blood plasma levels of the compound.
• Embodiment 1 An isolated fragment of PDE11 A4 comprising a GAF-B binding sequence and comprising or consisting of a polypeptide sequence of SEQ ID NO: 1, or a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.
• Embodiment 2 The isolated fragment of Embodiment 1, wherein the isolated fragment comprises and or consists of a polypeptide sequence of SEQ ID NO: 1.
• Embodiment s An isolated fragment of PDE11A4 comprising a GAF-B binding sequence and comprising or consisting of a polypeptide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.
• Embodiment 4 The isolated fragment of Embodiment 3, wherein the isolated fragment comprises and or consists of a polypeptide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
• Embodiment 5 A polynucleotide encoding an isolated fragment of PDE11A4 comprising a GAF-B binding sequence having an polypeptide sequence of SEQ ID NO: 1, or a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.
• Embodiment 6 A polynucleotide encoding an isolated fragment of PDE11A4 comprising a GAF-B binding sequence having an polypeptide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, or a sequence having at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 87%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity thereto.
• Embodiment 7 A vector comprising the nucleic acid of Embodiment 5 or
• Embodiment 8 A host cell comprising the polynucleotide of Embodiment 7.
• Embodiment 9 The host cell of Embodiment 8, wherein the cell is a mammalian cell.
• Embodiment 10 The host cell of Embodiment 9, wherein said host cell is a HT-
• Embodiment 11 A pharmaceutical composition comprising the isolated fragment of any one of Embodiment 1-4, and a physiologically compatible carrier medium.
• Embodiment 12 A pharmaceutical composition comprising the isolated fragment of any one of Embodiment 1-4, and a physiologically compatible earner medium, wherein the amount of the isolated fragment in the composition is a therapeutically effective amount for the treatment or prevention of a disease or disorder alleviated by inhibiting PDE11 A4 activity in a patient in need thereof.
• Embodiment 13 The pharmaceutical composition of Embodiment 11 or Embodiment 12, wherein the disease or disorder is associated with cognitive decline.
• Embodiment 14 The pharmaceutical composition of Embodiment 13, wherein the disease or disorder is selected from dementia, Alzheimer’s Disease (AD) including mild Alzheimer's disease and early-onset Alzheimer’s disease, Down’s syndrome, vascular dementia (cerebral amyloid angiopathy and stroke), dementia with Lewy bodies, HIV dementia, Mild Cognitive Impairment (MCI); Age-Associated Memory Impairment (AAMI); Age-Related Cognitive Decline (ARCD) (including age-related cognitive decline of associative long-term memories (aLTMs), dementia associated with traumatic brain injury, preclinical Alzheimer's Disease (PC AD); Cognitive Impairment No Dementia (CIND), and cognitive decline associate with spatial memory, other depression-related behaviors, additional anxiety-related behaviors, sensorimotor gating, and social behaviors.
• AD Alzheimer’s Disease
• MCI Mild Cognitive Impairment
• AAMI Age-Associated Memory Impairment
• ARCD Age-Related Cognitive Decline
• ALD Alzheimer-Related Cognitive Decline
• Embodiment 15 A method of treating or preventing a disease or disorder alleviated by inhibiting PDE11 A4 activity in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of the isolated fragment of any one of Embodiment 1 -4.
• Embodiment 16 A method of treating or preventing a disease or disorder alleviated by inhibiting PDE11 A4 activity in a patient in need thereof, comprising administering to the patient a therapeutically effective amount of a pharmaceutical composition of any one of Embodiment 11-14.
• Embodiment 17 The method of any one of Embodiment 15 or Embodiment 16, wherein the isolated fragment is administered in a dosage unit form.
• Embodiment 18 The method of Embodiment 17, wherein the dosage unit comprises a physiologically compatible carrier medium.
• Embodiment 19 The method of any one of Embodiment 15-18, wherein the disease or disorder is associated with cognitive decline.
• Embodiment 20 The method of Embodiment 19, wherein the disease or disorder is selected from dementia, Alzheimer’s Disease (AD) including mild Alzheimer's disease and early-onset Alzheimer’s disease, Down’s syndrome, vascular dementia (cerebral amyloid angiopathy and stroke), dementia with Lewy bodies, HIV dementia, Mild Cognitive Impairment (MCI); Age- Associated Memory Impairment (AAMI); Age-Related Cognitive Decline (ARCD) (including age-related cognitive decline of associative long-term memories (aLTMs), dementia associated with traumatic brain injury, preclinical Alzheimer's Disease (PCAD); Cognitive Impairment No Dementia (CIND), and cognitive decline associate with spatial memory , other depression-related behaviors, additional anxiety-related behaviors, sensorimotor gating, and social behaviors.
• AD Alzheimer’s Disease
• MCI Mild Cognitive Impairment
• AAMI Age- Associated Memory Impairment
• ARCD Age-Related Cognitive Decline
• ALD Alzheimer-Related Cognitive Decline
• Example 1 Phosphodiesterase 11A (PDE11A) and its Role in the Neurobiological Substrates of Memory and Social Behaviors
• This Example describes data testing the hypothesis that age-related increases in HIPP PDE11A4 occur in a compartmentalized manner and impair social aLTMs.
• a novel conditional transgenic system that controls the expression of PDE11A4 in a time- and brain region-specific manner by combining overexpressing or knockdown lentiviruses with PDE11 A WT and KO mice is used.
• RNAscope probes are used to delineate nuclear vs. cytosolic localization of PDE11A4 mRNA.
• In vivo/ex vivo techniques are used to study of PDE compartmentalization — and its functional consequences — rather than the study of artificial FRET-based constructs in in vitro assays.
• Age-related cognitive decline is not a uniform process, with vanability in symptom severity observed across cognitive domains.
• Human studies have demonstrated that associative long-term memories (aLTMs) — particularly those involving experiences with family and friends — are more susceptible to age-related cognitive decline than are recognition long-term memories (rLTMs).
• aLTMs associative long-term memories
• rLTMs recognition long-term memories
• “young” is defined as 2-6 months of age and “Old” is defined as 18-22 months of age since 1) protective effects are observed in PDE1 1 A KO mice as early as 14 months old and 2) it is expected 50% of the colony dies by the age of 24 months (i.e., do not want to introduce a selection bias by preferentially studying older mice with exceptional longevity).
• PDE11A WT or KO mice are administered a lentivirus containing a control fluorescent protein (EmGFP, as appropriate) or an N-terminal EmGFP-tagged PDE11A4 construct.
• EmGFP control fluorescent protein
• Viral vectors were delivered using stereotaxic techniques similar to those that have been described previously, except that inj ections were made directly into the hippocampus. Coordinates were selected to target CAI and subiculum — the portions of the hippocampus that naturally express PDE11A4 (DHIPP coordinates: AP -1.7 mm, ML +/- 1.6 mm, DV -1.4 mm; VHIPP coordinates: AP -3.3 mm, ML +/- 3.5 mm, DV -4.4 mm).
• the ability to virally manipulate PDE11 A4 expression has been measured and function in vivo and to measure aLTM and rLTM in virally -treated mice (Figs. 5A-5E).
• adolescent and young adult PDE11 A KO mice show intact STM and remote LTM (Figs. 4A-4B and 5A-5E), but impaired recent LTM for social experiences.
• the studies to date indicate that PDE11 A KO mice exhibit transient amnesia by virtue of expediting systems consolidation, which temporanly “misplaces” the memory but ultimately results in a strengthened memory trace in the cortex. While it remains to be determined if old PDE11A KO mice similarly show a form of transient amnesia (intact STM, impaired recent LTM, improved remote LTM), such an effect on systems consolidation could explain why old KOs are protected from age-related cognitive decline (see below for Arc mapping study that tests for this possibility).
• cannula tracks and PDE11 A4 expression levels are verified in each virally -treated subject. If a viral delivery is determined to have failed (e.g., cannula tracks miss the hippocampus, PDE11A4 expression not changed) data from that subject are dropped. Approximately equal numbers of male and female offspring are used in all studies (see infra for specific n’s). All experiments are counterbalanced for sex and genotype, but data are collected by an experimenter blind to genotype. Results
• Intramolecular signals have been identified that alter the trafficking of PDE11 A4, including homodimerization and N-terminal phosphorylation (Figs. 7A-7G). As such, it is important to determine 1) if the rate/nature of PDE11A4 post-translational modifications change with age and, as a result, 2) if age-related increases in PDE11 A4 expression occur in a compartment-specific manner (i.e., if excessive PDE11A4 is aberrantly trafficked).
• a preliminary biochemical fractionation experiment also points to compartmentspecific effects.
• Age-related increases in VHIPP PDE11 A4 occur primarily in the membrane fraction as opposed to the cytosol or nucleus, suggesting a mislocalization of the overexpressed PDE11 A4 (Fig. 9).
• this mislocalization is likely driven by the increase in PDE11 A4-pSl 17/pSl 24 because the in vitro studies show that S117D/S124D shifts PDE11A4 from the cytosol to the membrane (Fig. 7B).
• disrupting PDE11A4 homodimerization reduces PDE11A4 accumulation and shifts PDE11 A4 from the membrane back to the cytosol. Importantly, disrupting PDE11A4 homodimerization also reduces PDE11A4 cGMP hydrolytic (Fig 7G), consistent with the fact that it reduces pSl 17/pS124-PDEl 1A4 (Fig 7D). As such, studies here determines if preventing phosphorylation at S117/S124 or disrupting homodimerization are sufficient to prevent/reverse 1) age-related accumulation ofPDEHA4 in filamentous structures and/or the membrane and 2) age-related deficits in social aLTMs.
• mice and rats are available as live animals and, thus, are tested for remote LTM of STFP, SOR, and NSOR to confirm a selective age-related cognitive decline of social aLTMs.
• Monkey tissue is available only from post mortem stock and, thus, subjects are not cognitively phenotyped.
• age-related expression changes in PDE11A4 do appear to be conserved between rodents and primates at the level of total FIIPP mRNA levels (rat:; human: Fig. 2B); this is verified at the level of PDE11A4 protein expression and compartmentalization.
• Tissue from PDE11 A KO mice are processed in parallel as a negative control.
• changes in CAI dendrites of stratum radiatum proximal to the cell body indicates modulation of CA3 input signals
• changes in distal CAI dendrites indicates modulation of entorhinal cortex input signals.
• the age-related increases in PDE11 A4 expression most stnkmgly occur in the filamentous structures; however, the preliminary study also showed that a subset of sporadically distributed neurons in the VHIPP stratum pyramidale exhibit increased PDE11 A4 expression around the cell body. The sporadic nature of these cell bodies raises the possibility that these neurons reflect either a specific subtype of inhibitory interneuron or neurons that send projections to a discrete brain region.
• PDE11 A4 continues to only be expressed in excitatory neurons during aging, then retrograde tracer studies using stereotaxically-delivered fluorogold are conducted to determine if this neuronal subpopulation segregates based on their projections.
• anterior cingulate cortex, entorhinal cortex, and retrosplenial cortex as these brain regions show heightened activation in the PDE11 A KO during retrieval of enhanced remote social aLTM.
• Nucleus accumbens are also of interest given that a subset of PDE11 A4-expressing neurons project to NAcc and ventral CAI -NAcc projections are required for social aLTM.
• Defining changes at the level of subcellular domain To determine which subcellular domain constitutes the “filamentous structures” in which PDE11A4 accumulates with age, the slides collected above along are used with the previously published techniques to co-label for PDE11A4 and vanous markers, including those for axons, dendntes, gha, perineuronal nets, collagen, etc. Biochemical fractionation is conducted using DHIPP and VHIPP dissected from the second hemisphere of the rodents as well as anterior and posterior HIPP tissue samples from primates, with resulting fractions run on denaturing or native Western blots, all as per the published techniques .
• the isolated GAF-B domain (or control) are chronically expressed via lentiviral injection to the HIPP of old PDE11 A WT mice to determine if disrupting PDE11A4 homodimerization is sufficient to restore aged PDE11A4 phosphorylation, expression, and trafficking patterns to those observed in young mice and, in so doing, prevent age-related decline of social aLTMs.
• the lentiviruses express for at least 3 months, allowing for chronic manipulations.
• the GAB-B lentivirus have been obtained and it has been confirmed that it expresses in vivo.
• the effects of virally overexpressing S117A/S124A vs PDE11A4 WT (VS.
• a control lentivirus are compared in HIPP of old PDE11A KO mice. In so doing, it is determined if preventing phosphorylation of SI 17/S124 is sufficient to block the accumulation of PDE11A4 that is seen with high levels of endogenous PDE11A4 expression (Fig. 8) or viral overexpression of PDE11A4 W/T Social aLTM using STFP is also assessed to determine if preventing phosphorylation of SI 17/S124 blocks the ability of PDE11A4 overexpression to impair social aLTM (Figs. 5A-5E). Together, these studies help understand how/why age-related increases in PDE11A4 lead to age-related decline of social aLTM and, in so doing, identify novel therapeutic mechanisms by which the ectopic localization of PDE11A4 that occurs with age is addressed.
• tissue is labeled from EmGFP-PDEl 1 A4 infected PDE11 A KOs since overexpressed EmGFP- PDE11A4 also accumulates in filamentous structures in KOs and has been previously validated a GFP antibody in EM using EmGFP-PDEl 1A4 transfected COS-1 cells.
• Primates may show PDE11A4 expression in additional/ alternative hippocampal subfields (e.g., DG) or cell types (e.g., inhibitory interneurons) than mice and rats, which is important given that location infers function.
• additional/ alternative hippocampal subfields e.g., DG
• cell types e.g., inhibitory interneurons
• the functional consequences of virally expressing PDE11A WT vs. a control virus in those additional subfields or specific cell types (i.e., by using a cell type-specific promoter) using WT mice are interrogated.
• Identify molecular mechanisms driving age-related increases in PDE11A4 expression [00219] As described above, the spatially restricted nature of PDE11 A4 expression is maintained across the lifespan; however, steady-state levels of PDE11A4 protein expression within the HIPP steadily increase.
• PDE11A4 protein expression is also increased in the aged rat brain and more recently discovered that PDE1 1 A mRNA increases with age in the human hippocampus (Fig. 2B).
• age-related increases in PDE11A4 protein expression appear to be driven, at least in part, by increases in PDE11A4 transcription and/or transcript stability.
• transcription of a given gene is controlled by a core promoter, promoter-proximal elements, as well as enhancers or silencers.
• the core promoter falls within 30 base pairs (bp) of the transcription initiation site (TIS) and the promoter- proximal elements fall within 200 bp of the TIS, enhancers and silencers can fall anywhere within 50 kB of the TIS.
• TIS transcription initiation site
• enhancers and silencers can fall anywhere within 50 kB of the TIS.
• hPDEl 1 A4, hPDEl 1 A3, and hPDEl 1 Al in the 1200 bps upstream of their respective TISs.
• a lentivirus construct that uses the 1200 bps upstream of the mPdella4 TIS to drive expression of the mCherry reporter (/w/V/cA/aAmCherry) was developed.
• the decision to take a lentiviral approach is based on a prototype study by Chhatwal and colleagues.
• XRN2 In situ hybridization and Western blots are conducted on young vs. old HIPP tissue collected as described above to determine if age-related decreases in p54 mb /NONO and XRN2 are observed in rodents and rhesus monkeys as they are in humans (Fig. 11). Immunoprecipitation are also conducted using total homogenates using methods described above. Antibodies are used against p54 Iub /NONO and XRN2 to perform pull downs and then RT-PCR is conducted for PDE11 A4 mRNA. It is determined if old animals show less binding of pSd ⁇ /NONO and XRN2 to the PDE11 A4 transcript than do young animals.
• RNAscope probes enable single molecule-level resolution (see Fig. 12A) and when combined with confocal imaging (Fig 12B), enable the qualitative assessment of PDE11 A mRNA expression in the nucleus vs. the cytosol.
• the finding that aging is associated with increased PDE11 A4 transcription or transcript stability does not rule out the possibility that aging is also associated with increased rates of translation or protein stability, and the possibility of age-related reductions in sumoylation or ubiquitination of PDE11A4 is further examined.
• the pilot data suggests the 1200 bps upstream of the PDE11 A4 TIS is sufficient to control the transcription of mPdella4 in terms of its spatial distribution, but it is possible that aging also/altematively could influence rates of transcription by differentially engaging enhancer or silencing motifs within 50 kB of the TIS.
• BAC transgenic mice are generated that express a fluorescent protein under the control of a minimal promoter coupled with various combinations of PDE11A4 exons, introns, 5’ upstream sequences, and 3’ downstream sequences.
• exoribonucleases are not the only mechanism regulating transcript stability; microRNAs, along with other noncoding RNAs, play an important role as well.
• the focus is to identify non-coding RNAs that regulate PDE11 A4 expression.
• miR-375 is one of only 4 microRNAs that is predicted by TargetScan to target the PDE11A4 transcript.
• miR-375 expression decreases with age in the mouse brain, which is consistent with the observed age- related increases in PDE11A4 .
• data are analyzed by multifactorial ANOVAs or by repeated measure ANOVAs where appropriate to account for multiple comparisons.
• in vivo data are analyzed for effects of sex, genotype, lentiviral treatment (in addition to assay-specific factors, such as food type).
• Statistical outliers are dropped from analyses, as previously described.
• Significant ANOVAs are followed by Student Newman-Keuls post hoc tests, with significance determined as P ⁇ 0.05.
• Data in figures are plotted as means ⁇ SEMs.
• mice are genotyped a priori to enable proper counterbalancing of experimental run lists; however, experimenters are blind to genotype at the time of data collection. Genotypes are then reconfirmed post death by Western blot or in situ hybridization. Physical parameters are counterbalanced across subjects (e.g., which is the “trained” spice and which is “novel”). Biological Variables: both males and females were tested for effect of sex.
• PDE1 1 A represents a therapeutic target not only for preventing age-related decline of social aLTMs but also for reversing deficits.
• the effects of excessive PDE11A4 on acquisition vs. consolidation, vs. retrieval of remote social aLTMs are studied as well as those aimed at understanding the system-level mechanism by which altered cyclic nucleotide signaling in the VHIPP can impair social aLTMs (e.g., by compromising the integrity of neuronal ensembles encoding the memory engram in the hippocampus vs.
• PDE11 A4 is problematic simply by virtue of increased steady state levels that are ubiquitously distributed or by virtue of a discretely localized upregulation or even an ectopic expression of PDE11A4.
• the effect of modulating PDE11A4 function within specific cell-types (e.g., excitatory vs. inhibitory) or sub-region (e.g., subiculum vs. CAI) is examined.
• the upstream signaling events are delineated, including the specific kinases that lead to age-related increases in the phosphorylation of PDE11 A4.
• PDE11 A is a highly druggable enzyme and it is positioned to selectively control cyclic nucleotide signaling in a molecularly-defined circuit that specifically regulates social LTMs, without affecting signaling elsewhere. This may relieve age-related impairments in aLTM without causing unwanted side effects.
• PDE11A exhibits all properties the pharmaceutical industry believes an ‘ideal’ drug target should have. The fact that PDE11 A is a realistic candidate for drug development increases the value of understanding its biological function.
• this pool of accumulated PDE11 A4 is uniquely phosphorylated at serines 117 and 124.
• phosphomimic mutation of SI 17 and S124 drives accumulation of PDE11A4, while phosphoresistant mutations or blocking homodimerization reduces this accumulation.
• This Example describes studies demonstrating that age-related increases in phosphodiesterase 11A (PDE11A), an enzyme that degrades 3’,5’-cAMP/cGMP and is enriched in the ventral hippocampal formation (VHIPP), drive age-related cognitive decline (ARCD) of social memories.
• PDE11A phosphodiesterase 11A
• VHIPP ventral hippocampal formation
• ARCD drive age-related cognitive decline
• age-related increases in PDE11 A4 occur specifically within the membrane compartment and ectopically accumulate in filamentous structures termed ghost axons.
• Previous in vitro studies show that disrupting PDE11 homodimerization by expressing an isolated PDE11A-GAFB domain that acts as a “negative sink” for monomers selectively degrades membrane-associated PDE11 A4 and prevents the punctate accumulation of PDE11A4.
• C57BL6/J mice were originally obtained from Jackson Laboratory (Bar Harbor, ME) and the line was maintained at the University of South Carolina.
• the Pdel la mouse line obtained from Deltagen (San Mateo, CA) was maintained on a mixed C57BL6 background (99.8% multiple C57BL/6 substrains, 0.2% 129P2/01aHsd).
• Pdel la mice were bred at the University of South Carolina in heterozygous (HT) x HT tno-matings. Same-sex wild-type (WT), heterozygous (HT), and knockout (KO) littermates were weaned and caged together to total 3-5 mice/cage.
• mice normally contributes only 1-2 mice/genotype and parents contribute two litters at most to a cohort. While both males and females were used in experiments, analyze for sex effects (see figure legends for specific n’s/sex/group/experiment). In these studies, young mice were defined as 2-6 months and old mice were defined as 18-22 months.
• mice included both young Pdel la WT mice surgenzed alongside old Pdel la WT mice (i.e., receiving bilateral injections of mCherry lentivirus to the dorsal and ventral hippocampi) and unsurgerized young C57BL6/J mice that were used as an internal control for the assays (Figs. 14A-14B). Since no obvious differences were found between groups of young surgerized Pdel la WT mice and young unsurgerized C57BL6/J mice, the data from these 2 subgroups were subsequently combined into a singular “young” (Figs. 14C-14G). All mice used in experiments were generally healthy throughout the duration of testing. Gross pathology was not conducted but mice were routinely assessed by husbandry, veterinary, and laboratory staff. Mice with lethargy, altered gait, signs of malnutrition or dehydration, noticeable tumors >1 cm, were removed from study and euthanized.
• Tissue Collection Mice were euthanized (during light cycle) via rapid cervical dislocation and brains were immediately collected and flash frozen on 2-methylbutane sitting on dry ice. Brain tissue was then stored at -80 °C until cryosectioning at 20 pm to verify viral expression.
• the “demonstrator mouse” was returned to the original home cage where the “observer” cage mates were allowed unrestricted access to the demonstrator for 15 minutes. It is during this time that the observers make an association between the social pheromones in the breath of the demonstrator and the non-social odor (household spice). Recent and remote long-term memory were assessed 24 hours or 7 days after training, respectively. At that time, the observer mice were individually placed in clean home cages and given access to two flavored/powdered chows for 1 hour. One flavored chow contained a novel spice and the other contained the spice that the demonstrator was given. The amount of food eaten was measured by an experimenter blind to treatment.
• mice had to meet minimum inclusion criteria including eating at least 0.25 grams of food. Cohorts were able to be trained/tested at multiple time points using different spice combination to reduce the total number of mice used and we have shown that this does not confound interpretation of the data. Observer mice eating more food containing the familiar spice (i.e., the spice on their demonstrator’s breath) versus the novel spiced food constituted memory (preference ratio: familiarnovel/ famili ar+novel) .
• Training for SOR and NSOR consisted of a habituation trial with 3 beads from the subject’s home cage, followed by two training trials that included 2 home-cage beads and 1 novel-scented bead. Recent and remote long-term memory were assessed 24 hours or 7 days after training, respectively.
• mice were tested with one home cage bead, one bead from the trained donor strain (familiar), and one bead from a second donor strain (novel).
• mice were tested with only two beads, one scented with the training spice and one a novel spice. The designation of which scent was “novel” within a given testing trial and the location of the novel scent (i.e., left versus right) was counterbalanced across subjects.
• mice were given two minutes to investigate the beads and the amount of time spent on each was manually scored by an experimenter blind to treatment and bead. It was previously determined that infusion of even a negative control lentivirus into the hippocampus reverses the recent long-term memory impairment observed in Pdel la KO mice 24 hours after training. Therefore, 24-hour memory following injection of the isolated GAF-B domain was not tested as the results would not be interpretable. All mice met minimum inclusion criteria of spending a minimum of 3 seconds in total sniffing the beads. Spending more time investigating the novel vs familiar scent constituted memory (preference ratio: novel-familiar/novel+familiar). [00242] Stereotaxic Surgeries.
• Stereotaxic surgeries and injections were performed using a NeuroStar motorized stereotaxic, drill, and injection robot (Tubingen, Germany). Mice were anesthetized with a steady flow of oxygen and isoflurane. The mice were induced at 3% isoflurane and maintained at 1-1.5%. Lack of reflexes was verified and the scalp was then shaved and cleaned with betadine. A small incision was made in the scalp and the skull was cleared with sterile saline. Cotton swabs were again used to visualize the skull and locate Bregma.
• a Hamilton syringe custom needle #7804-04: 26s gauge, 1” length, 25 degree bevel was then then placed to the following depths relative to Bregma: dCAl DV, - 1.3, vCAl DV, -4.4.
• the injection robot was used to inject 2 pl of lentivirus at 0. 167 pl/minute. Following injection completion, the experimenter waited two minutes to allow the lentivirus to diffuse away from the needle and the needle was raised at the same speed. After all injections were complete, pronged tweezers were used to close the scalp and secured using glutures. Buprenorphine in sterile saline at a dose of 0. 1 mg/kg was injected IP for pain management. For recovery, the mouse was placed on a warm Deltaphase pad and allowed to recover until moving normally and posturing upright. Mice were allowed at least 2 weeks of recovery in grouped home cages prior to behavioral testing.
• a lentivirus carrying an mCherry -tagged PDE11 A4-GAFB served to disrupt PDE11 A4 homodimerization, while an mCherry-only virus was used as a negative control.
• a lentiviral construct was used here in order to compare to previous studies examining the effects of overexpressing PDE11 A4 in vivo, which required the use of a lentiviral cassette due to the large size of PDE11A4.
• the viruses were made on an “SPW” backbone that drives expression using the phosphoglycerate kinase 1 (PGK) promoter, which in theory is a ubiquitous promoter and yet is taken up preferentially by neurons.
• PGK phosphoglycerate kinase 1
• the isolated GAF-B construct disrupts PDE11A4 homodimerization by binding to PDE11A4 and triggering proteolytic degradation. For reasons that are not well understood, the GAF-B construct degrades PDE11A4 more significantly in the membrane versus cytosolic fractions.
• the lentiviruses were prepared and diluted in 0.2 M sucrose/42 mM NaCl/0.84 mM KC1/2.5 mM Na2HPO4/0.46 mM KH2PO4/0.35 mM EDTA and the original titers were as follows: mCherry, 7.37X10E10/mL; GAF-B, 1.82X10E10/ml.
• Multiple PDE11A antibodies were utilized to discern diffuse expression versus accumulations of PDE11 A4. While the PDE11A4#1 antibody labels all PDE11A4 and, thus, detects both diffusely localized and accumulated PDE11A, the PDE11A4-140 and 150 antibodies specifically labels PDE11A4 phosphorylated at serines 117 and/or 124 and, thus, only detects PDE11A4 accumulated in ghost axons.
• mCherry antibodies hosted in a rodent species were pretreated with anti-Mouse FabFragments (0.15mg/ml; Jackson Immunoresearch # 715-007-003) in PBS for 2 hours, followed by 3x10 minute washes in PBT prior to adding primary antibody.
• Primary antibody solution was added over brain sections and the slides were kept level at 4 °C overnight. Primary antibodies was removed using 4x10 minute washes in PBT.
• PDE11A4 secondary antibody Alexafluor 488 AffiniPure Donkey Anti-Chicken, 1 : 1000, Jackson Immunoresearch #703- 545-155
• the secondary was washed off using 3X10 minute washes with PBT.
• the mCherry secondary Al exafluor 594 AffiniPure species-specific, 1 : 1000, Jackson Immunoresearch
• 3x10 minute washes with PBT were used to clear the slides of any remaining secondary and the slides were briefly dip-rinsed in PBS to remove Triton.
• PDEl lA4-filled structures were quantified by an experimenter blind to treatment with images captured using Leica Application Suite (LASX) software and a Leica DM5000 B florescent microscope. Brightness, histogram stretch, and/or contrast of images was adjusted for graphical clarity .
• LASX Leica Application Suite
• COS-1 male line
• HEK293T female line
• HT-22 sex undefined cell culture and transfection were performed as previously described [1] While kept in t-75 flasks, cells were grown in Dulbecco’s Modified Eagle Medium (DMEM) (GIBCO; Gaithersburg, MD, USA), 10% fetal bovine serum (FBS) (GE Healthcare Life Sciences; Logan, UT, USA), and 1% Penicillin/Streptomycin (P/S) (Mediatech, a Coming subsidary; Manassas, VA, USA).
• DMEM Modified Eagle Medium
• FBS fetal bovine serum
• P/S Penicillin/Streptomycin
• Biochemical Fractionation and Western Blotting Biochemical Fractionation was performed to obtain cytosolic and soluble membrane fractions. Cell were mixed with ice cold fractionation buffer (20 mM Tris-HCl, pH 7.5; 2 mM MgC12; Thermo Pierce Scientific phosphatase tablet A32959 and protease inhibitor 3 #P0044) and sonicated.
• a low- speed spin 1000 x g removed cellular debris and the supernatant from this spin was transferred to a new tube.
• a high speed spin 89,000 x g was performed to obtain the membrane (pellet) and cytosolic (supernatant) proteins.
• the membrane pellet was sonicated and subjected to a high-speed spin (60,000 x g). The supernatant containing all membrane proteins was then placed in a new tube. Samples were nutated for at 4 °C for 30 minutes.
• a second high-speed spin (60,000 x g) was done for 30 minutes to separate the soluble membrane (supernatant) from the insoluble membrane (pellet). The soluble membrane sample was then transferred to a clean tube and used for western blot (see below).
• a DC Protein Assay kit (Bio-Rad; Hercules, CA, USA) was used to determine protein concentrations by which total protein was equalized across samples. Samples were stored at - 80 °C until used in Western blotting. For Western blotting, 10 ug of protein was loaded onto 12% NuPAGE Bis-Tris gels (Invitrogen, Waltham MA) and run at 180 volts.
• PDE assay cAMP- and cGMP-PDE catalytic activity were measured. The assay was validated in vitro using HT-22 cells (a mouse hippocampal cell line). Buffer containing 20 mM Tris-HCl and 10 mM MgC12 was used to harvest cells and kept on ice until ready to use. PDE activity was measured using 50 pl of sample and 50 uL of [3H] cAMP (Perkin Elmer, NET275) or cGMP (Perkin Elmer, NET337) and incubated for 10 minutes. After incubation, 0. IM HC1 was added to quench the reaction, followed by 0.1M Tris to neutralize the reaction.
• Disrupting PDE11 A4 homodimerization selectively decreases PDE11A4 expression in a compartment-specific manner and is sufficient to decrease PDE11 A4 accumulations in ghost axons that occur with aging. It was previously found that ventral hippocampal PDE11 A4 expression increases with age in both mice and humans and that these age-related increases accumulate specifically in the membrane compartment and within filamentous structures termed “ghost axons”. Additionally, it was found in vitro that disrupting PDE1 1 A4 homodimerization by expressing an isolated GAF-B binding domain that acts as a negative sink (Fig. 13B) leads to proteolytic degradation specifically of membrane-bound PDE11A4 and reduces the accumulation of PDE11A4 into punctate structures.
• lentiviruses were used (Fig. 13A) containing either mCherry alone (i.e., negative control) or an mCherry -tagged isolated GAF-B domain that disrupts PDE11A4 homodimerization (Fig. 13B). These lentiviruses were stereotactically injected bilaterally into the CAI field of dorsal and ventral hippocampi of old Pdel la WT mice, since this is the field where PDE1 1 A4 regulates social learning and memory.
• mice demonstrated mCherry signal in dorsal and ventral CAI, with a subset of mice exhibiting expression in neighboring hippocampal sub-regions (e.g., dentate gyrus, CA3, CA2) that do not express PDE11A4 (Fig. 13D). While mCherry -treated mice exhibit a uniform pattern of PDE11A4 expression across stratum radiatum, stratum pyramidale and stratum oriens of CAI, GAF-B treated mice exhibit a compartment-specific decrease in PDE11A expression, with reduced expression in the distal segment of stratum radiatum and stratum oriens relative to stratum pyramidale (Fig. 13E). Consistent with the fact that the isolated GAF-B domain reduces PDE11 A4 protein expression in the membrane compartment, it was found that disrupting homodimerization of PDE11A4 reduced age-related increases in so-called
• PDE11 A4 ghost axons i.e., filamentous structures harboring age-related accumulations of PDE11A4.
• Fig. 13F The ability of the GAF-B construct to reduce the accumulation of PDE11 A4 in ghost axons was confirmed using two different PDE11 A4 antibodies on two different sets of slides (Fig. 13G). Without wishing to be bound by any particular theory, all together these data suggest disruption of PDE11A homodimerization in vivo is sufficient to reverse age-related increases in PDE11A4 protein expression and ectopic accumulation. [00252] Disrupting PDE11 A4 homodimerization in the hippocampus of old mice is sufficient to reverse age-related decline of remote long-term social memory.
• disrupting PDE11A4 homodimenzation like genetically deleting PDE11A — does not alter the ability to detect, leam about, or retrieve memories for recognizing non- social odors.
• disrupting PDE11A4 homodimerization again, like genetically deleting PDE11A — did significantly improve remote LTM for SOR memory (Fig. 14G; Table 1), despite having no effect on SOR learning (Fig. 14F) or total time sniffing (Table 2).
• these data suggest that disrupting PDE11A4 homodimerization is sufficient to reverse ARCD of remote social memory .
• PDE11 A4 homodimerization is an independent intramolecular mechanism that regulates PDE11 A4 trafficking and functioning. As noted above, disrupting PDE11 A4 homodimerization significantly changes the subcellular compartmentalization of the enzyme. To better understand the functional consequences of disrupting PDE11 A4 homodimerization, we measured PDE activity and cyclic nucleotide levels in cells transfected with GFP + mCherry (i.e., negative control), GFP-PDE11A4 + mCherry, or GFP-PDE11A4 + mCherry - GAF-B.
• GFP + mCherry i.e., negative control
• GFP-PDE11A4 + mCherry i.e., negative control
• GFP-PDE11A4 + mCherry i.e., negative control
• GFP-PDE11A4 + mCherry i.e., negative control
• the ability of the GAF-B construct to alter cyclic nucleotide levels in absence of a direct effect on PDE11 A4 enzymatic activity is likely due to changes in subcellular compartmentalization of PDE11A4 possibly shifting it from a cGMP-rich pool to a cAMP-rich pool.
• disrupting homodimerization not only reduces the accumulation of WT PDE11A4, it also reduces back to WT levels the potentiated accumulation caused by the aging-related phosphomimic mutant SI 17D/S124D .
• the isolated GAF-B domain could reduce accumulation not only of WT PDE11 A4 but also PDE11 A4-S117D/S124D, which mimics the age-related increase in PDEl lA4-pS117/pS124 that drives the punctate accumulation of PDE11A4 in the aged brain.
• the isolated GAF-B domain reduced the punctate accumulation of both the WT and PDE11 A4- S117D/S124D compared to the mCherry control (Fig. 6E-6F).
• S162D changed the subcellular compartmentalization of PDE11 A4 in a manner similar to that observed with the isolated GAF-B domain — namely, shifting PDE11 A4 from the membrane to the cytosolic fraction (Fig. 6G).
• the isolated GAF- B domain was able to effectively reduce the accumulation of both WT PDE11 A4 as well as the phosphoresistant PDE11A4-S162A , suggesting phosphorylation of S162 is not needed for the dispersing effect of the isolated GAF-B domain (Fig. 6H-6I). While not wishing be bound by any particular theory, this result suggests that disrupting homodimerization does not achieve effects by promoting phosphorylation of SI 62.
• S162D differed substantially from GAF-B in terms of regulating cyclic nucleotide levels. It was found that S162D elicited quite different effects on cyclic nucleotide levels than were described above for the isolated GAF-B domain (Figs. 6C-6D). Specifically, S162D did not alter PDE11 A4 hydrolysis of cGMP (Fig. 6J) and appeared to reduce PDE11A4 hydrolysis of cAMP (Fig. 6K). Although not wishing to be bound by any particular theory , together these data suggest that homodimerization and pS162 are independent intramolecular mechanism that regulate PDE11A4 trafficking and function.
• disrupting PDE11A4 homodimerization within hippocampal CAI using a biologic encoding an isolated GAF-B domain reverses the age- related accumulation of PDE11 A4 in ghost axons in vivo, reverses the accumulating effect of SI 17D/S124D in vitro, and reduces PDE11A4 expression in ventral CAI in a compartmentspecific manner (i.e., in distal dendrites and stratum oriens; Figs. 13E-13G).
• disrupting PDE1 1 A homodimerization is sufficient to reverse ARCD of remote social aLTMs — albeit at the expense of an inability to retrieve recent social LTMs (Figs. 14A-14G ).
• PDE11A4 Post-translational modifications and protein-protein interactions alter the subcellular localization and trafficking of PDE11A4 protein.
• the subcellular compartmentalization of PDEs are regulated by multiple mechanisms.
• the localization of the PDE2, PDE4, PDE5, PDE10, and PDE11 families is regulated in part by post-translational modifications.
• phosphorylation of PDE10A prevents membrane insertion by blocking palmitoylation.
• PDE11A4 is thought to associate with the membrane by binding to a macromolecular complex.
• the ability of PDE11 A4 to interact with this macromolecular complex appears to be reduced when PDE11A4 homodimerization is disrupted or when S162 is phosphorylated (Fig.
• PKA and PKG have been shown to phosphorylate SI 17 and SI 62 in vitro, and these same kinases are predicted to phosphorylate SI 24. Cyclic nucleotide levels controlled by PDE11A4 could then modulate PKA/PKG activity at SI 17, SI 24, and/or SI 62, thereby creating direct feedback/feedforward loop as has been described for other PDE families.
• This A499T BALB/cJ mutation strengthens homodimerization, elevates protein expression, and increases the punctate accumulation of PDE11A4 both in vitro and in vivo.
• disrupting homodimerization using the isolated GAF-B domain has the opposite effects, lowering protein expression of PDE11A4 due to increased proteolysis and reducing the punctate accumulation of PDE11A4 both in vivo and in vitro; Figs. 13E-13G, 6E-6F and 6H-6I).
• Disrupting PDE11 A4 homodimerization as a therapeutic approach for treating ARCD A hallmark pathology of the aging brain includes ectopic protein expression and accumulation in the brain, as well as the loss of cGMP signaling in the hippocampus.
• disrupting PDE11 A4 homodimerization significantly decreases the puctate accumulation of PDE11A4 and specifically promotes degradation of membrane-associated PDE11 A4.
• the results described herein suggest that disruption of PDE1 1 A4 homodimerization may be a sophisticated mechanism for therapeutic targeting of age-related increases in PDE11A4, by decreasing both its accumulation and clearing it from select compartments.
• the data described herein demonstrate that disruption of PDE11A4 homodimerization using a biologic — such as the biologic described herein — represents a novel therapeutic approach for treating ARCD, including ARCD of social memories.
• Phosphodiesterase 11A in brain is enriched in ventral hippocampus and deletion causes psychiatric disease-related phenotypes. Proceedings of the National Academy of Sciences of the United States of America. 2010,107(18):8457-62. doi: 10. 1073/pnas. 1000730107. PubMed PMID: 20404172; PubMed Central PMCID: PMC2889541.
• PDE9A new phosphodiesterase 9A
• Chhatwal JP Hammack SE, Jasnow AM, Rainnie DG, Ressler KJ. Identification of cell-type-specific promoters within the brain using lentiviral vectors. Gene therapy.
• Gaveriaux-Ruff C Kieffer BL.
• Conditional gene targeting in the mouse nervous system Insights into brain function and diseases. Pharmacology & therapeutics.

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