Preliminary clinical data on Y 1 -targeted tumor imaging with the latter compound in breast cancer patients are encouraging [ 18 ]. There is no information yet on Y 1 receptor antagonists coupled to a chelator, which are suitable for in vivo receptor targeting. The aim of the present study was to design and develop dimeric NPY analogs coupled to the DOTA chelator for suitable radiolabeling that could be used for imaging and for radiotherapy of Y 1 -expressing tumors.
All reagents were of best grade available and were purchased from common suppliers. Silver trifluoromethanesulfonate was obtained from Sigma-Aldrich St. Structure of the dimeric NPY scaffold and of the incorporated substitutions. Then, they were incubated for min in the incubation solution containing the Krebs-Ringer solution, 0. Membrane pellet sections were incubated with I-hPYY in increasing concentrations ranging from 0.
After the incubation, the slides were washed two times for 5 min and then rinsed four times in ice-cold preincubation solution. IC 50 values were calculated after quantification of the data using a computer-assisted image processing system Analysis Imaging System, Interfocus, Mering, Germany.
Medium was then removed and replaced with fresh medium containing 0. After removal of the medium, cells were lysed and cAMP accumulation was determined using the SMP kit from PerkinElmer according to the instructions of the manufacturers. Xa Xa 29'. HPLC a. CZE b. Detection at nm.
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ND, not determined. All four tested compounds exhibit Y 1 selectivity. Intracellular cAMP accumulation was then determined as described in Methods. Compound 11 behaves like an antagonist since it shifts the dose response curve of LP-PYY to the right. The recent demonstration that radiolabeled antagonists considerably improved the sensitivity of in vivo diagnostic procedures and might improve the efficacy of receptor-mediated radiotherapy suggested the generalized use of peptide antagonists rather than agonists for in vivo tumor detection [ 5 , 30 ].
However, selective peptide antagonists suitable for radiolabeling are not available for each receptor candidate and therefore they need to be developed. The high density and incidence of Y 1 receptors in invasive and metastatic breast cancers, also expressing Y 2 but not Y 4 and Y 5 receptors, make these neoplasms important targets for diagnosis and therapy with NPY-related drugs [ 8 ]. Thus, the aim of the present study was to design Y 1 receptor affine antagonists suitable for molecular imaging. As demonstrated, NPY exists in equilibrium between monomer and dimers in aqueous solution [ 31 ].
These dimeric structures were found to be not mandatory for the binding of NPY to Y 2 receptors but were responsible for the increased Y 1 -affinity and selectivity. Proof of this concept was supported by the synthesis and the characterization of several dimeric NPY analogs retaining high Y 1 -affinity and selectivity over Y 2 [ 23 , 25 ]. We thus derived a conceptual and experimental approach to the design of new dimeric Y 1 receptor antagonists using the previously described dimeric scaffold, i.
As supported by a previous report, replacement of Tyr 32 by Trp and Leu 34 by Nle 3 did not produce a significant change in binding affinity or selectivity. However, adjunction of DOTA moieties to the N-termini of these dimeric peptides resulted in a dramatic loss of binding affinity 2 and 4 suggesting the importance of the N-terminus for receptor recognition. In accordance, evidence demonstrating that N-terminal acylation might compromise receptor affinity and selectivity were already reported in the literature [ 33 , 34 ].
Such observation prompted us to evaluate different sites for the introduction of the DOTA. However, such substitution in each segment of the peptide dimers 6 and 10 resulted in a loss of Y 1 receptor-binding affinity compared to their DOTA-free counterparts 5 and 9. Most likely, this reduction of affinity is probably due to the size and the negatively charged character of the DOTA moiety.
As observed by NMR in BVD15, a monomer segment analogous to our peptide dimer, the Arg 33 , which was found to be particularly involved in the Y 1 receptor recognition [ 27 ], and Asn 29 side chains seem to be spatially closed [ 35 ]. It is thus probable that coupling the DOTA moiety to the Dpr residue might disrupt the orientation or the character of the positively charged Arg residue and thus alter the overall binding affinity.
Supporting this hypothesis, increasing the distance between the DOTA moiety and the peptide backbone, i. The higher binding affinity of homodimeric NPY peptides was often related to the propensity of NPY receptors to form homodimers as recently demonstrated [ 36 ]. As such, by keeping intact one segment, original interaction between heterodimers 8 and 11 and their receptor was restored; these two compounds being almost equipotent compared to the lead precursor 1. Although preliminary in nature, our results, and more particularly those of analog 11 , represent the first step towards the development of dimeric DOTA-coupled Y 1 receptor antagonist for nuclear medicine application.
Even if the stability of these analogs has not been evaluated in the present study, these compounds should prove to be sufficiently stable for tumor targeting purpose. In analogy to somatostatin receptor targeting of tumors, it has been proposed to use NPY analogs to target NPY receptors for tumor therapy. NPY analogs suitable for this purpose have indeed already been developed, such as a daunorubicin-coupled cytotoxic NPY analogs [ 16 ] and a Y 2 -selective 99 m Tc-labeled radioactive NPY analog [ 17 ], all being agonists.
Assuming that the observation of the superiority of somatostatin receptor antagonists, but also of bombesin receptor antagonists [ 30 ] for tumor targeting can be generalized, the present DOTA-coupled high-affinity Y 1 receptor antagonist may be a useful tool for the diagnostic and radiotherapeutic targeting of Y 1 -expressing tumors. Breast tumors with their high Y 1 -receptor density would represent first choice candidate tumors. Other tumor types, such as renal cell carcinomas, ovarian cancers, adrenal tumors and embryonic tumors, may also be targets of interest. The same general principles as for somatostatin receptor targeting could be applied.
Advantages that should be put forward are a more favorable benefit-toxicity profile compared with conventional radio- or chemotherapy and the rarity of side effects. The radiotargeting of NPY receptor-expressing tumor blood vessels alone or together with NPY receptor-expressing tumor cells may also represent an attractive strategy for therapy. Finally, since many of the NPY receptor-expressing tumors can express multiple peptide receptors concomitantly, NPY receptors may be suitable for a multireceptor targeting with a cocktail containing NPY and other therapeutic peptide analogs directed against various peptide hormone receptors.
For such a multireceptor approach, good candidate tumors seem to be breast tumors targeted with NPY and bombesin analogs. In the course of designing and synthesizing DOTA-coupled dimeric NPY Y 1 receptor antagonists, we found that the addition of a DOTA moiety to both peptide segments negatively influenced the binding affinity of all dimeric compounds synthesized and that the asymmetric introduction of a DOTA to one of the segments of the peptide heterodimer yields compounds exhibiting high binding affinity and Y 1 -selectivity over Y 2. They may, when linked to an adequate radiometal, become useful tools for in vivo tumor targeting of Y 1 -positive tumors, particularly breast tumors.
This bountiful palette can permit the construction of personalized cancer therapeutics upon selecting a tumor-homing peptide that will be most appropriate for the type of cancer needed. In addition, peptide sequences can be selected according to the required physicochemical properties such as solubility, stability and overall charge or the characteristic groups necessary for the conjugation with the therapeutic payload.
The overall experimental procedure to synthesize a PDC is usually rapid and facile. Notably, the overall cost to produce a PDC, where an already approved drug can be selected and re-used from a pool of available cytotoxic agents, is much lower compared to the cost of synthesizing a new cytotoxic agent, as it is based on an already applied drug with the addition of a small peptide. Nevertheless, the last years more complex bioconjugates have been synthesized to allow the simultaneous diagnosis and therapy theranostics of diseases. The therapeutic efficacy of a PDC is predominantly associated with the potency of the drug and the targeting efficiency of the assembled conjugate.
Thus, PDCs should possess certain features to render them appealing candidates for treatment:. These peptides are cell-specific and bind to certain receptors promoting their internalization. They are usually inserted into the cell via endocytosis and then they are transported to intracellular compartments with higher concentration of enzymes and lower values of pH, where they disassociate from the receptor and afterward from the anticancer agent. The most representative examples of peptides utilized for PDCs are highlighted below. Linear peptides are included among the rich reservoir of options, finding applications in tumor targeting.
They exist in different lengths, structures and with various physicochemical properties. An immense number of cyclic peptides have been synthesized  and many of them have displayed superior affinity and selectivity for the receptor than their parent linear counterparts . Cyclic peptides are usually synthesized by reacting the N-terminus with the C-terminus or by exploiting specific functional groups of certain amino acids present in the sequence.
A representative example is the sulfhydryl group of cysteine-containing peptides which may cyclize through the formation of intramolecular disulfide bonds . The most commonly used linear peptides and cyclic peptides that can be delivered inside cancer cells via endocytosis and one that smuggles into glioma tissues via transcytosis angiopep-2 are presented below:.
The fact that carcinogenesis is highly dependent on migration, invasion and angiogenesis renders integrins important anticancer targets. This integrin is overexpressed on activated endothelial cells, new-born vessels and other tumor cells [48,49] , but it is found to be expressed at undetectable levels in most adult epithelial cells, making it a suitable target for anti-angiogenic therapy .
Gonadotropin-releasing hormone GnRH : Gonadotropin-releasing hormone GnRH , also known as luteinizing hormone-releasing hormone LHRH , is a hormone responsible for the secretion of two gonadotropins: follicle-stimulating hormone FSH and luteinizing hormone LH from the anterior pituitary gland. This peptide acts through a similar receptor type II GnRH-R , which is expressed in different tissues, including tumor cells.
GnRH-III binds to GnRH-R overexpressed on the cancer cell surface, resulting in an antiproliferative effect but seems to be less potent than the rest GnRH analogs regarding stimulating gonadotropin release at the pituitary level . GnRH peptide analogs constitute an emerging class of tumor homing peptides for malignant tissues expressing the GnRH-R. Their development is based on the fact that specific human cancer cells mostly ovarian, prostate, lung and breast uniquely express or overexpress GnRH-R with respect to normal tissues .
Therefore, covalent attachment of a cytotoxic agent to these peptides provides the possibility to produce potent tumor-targeting PDCs. Somatostatin SST : Somatostatin is a neuropeptide produced by neuroendocrine, inflammatory and immune cells and has an important role in various physiological functions acting as a classical endocrine hormone, a paracrine regulator or a neurotransmitter .
Both SST and SST exhibit biological activity through high-affinity membrane receptors somatostatin receptor 1—5; SSTR1—5 , that are widely distributed throughout the human body in various tissues like the nervous, pituitary, kidney, lung and immune cells [65,66]. SSTRs are overexpressed in various neuroendocrine malignant tumors NETs including pancreatic, pituitary, prostate, lung carcinoids, osteosarcoma etc. Therefore, these receptors can be targeted for selective delivery of efficient concentrations of cytotoxic warheads to the tumor sites.
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However, native somatostatin gets rapidly hydrolyzed due to enzymatic degradation and therefore, more stable and potent analogs have been developed. These analogs were synthesized by replacing L-amino acids with their D-isomers and reducing the length by keeping only the peptide epitope responsible for the biological activity. There are several examples of PDCs consisting of the aforementioned somatostatin targeting peptides [67,69,70] , as also other somatostatin peptide analogs, e.
EGFR is upregulated in a wide pool of cancer tissues and is able to enter cells usually via clathrin-mediated endocytosis . The BBB is formed by the endothelial cells of the brain, restricting and controlling the exchange of molecules between the central nervous system and the rest body.
Angiopep-2 is able to cross the BBB via receptor-mediated transcytosis after binding to the low-density lipoprotein receptor-related protein-1 LRP-1 , which is overexpressed in brain cells . Moreover, the two lysines available in its sequence render angiopep-2 an appealing PDC candidate, with the aim to smuggle therapeutic payloads to brain malignancies [78,79]. Cyclic peptide variants have been developed for the RGD peptide motif, reported above.
Figure 3: Binding and penetration mechanism of iRGD. The RGD motif is responsible for binding to integrins. The CendR element then interferes with the binding to neuropilin-1, resulting in tissue and cell penetration. The tumor-penetrating peptide can be used to decorate a cargo a simple chemical moiety or a nanoparticle , but only in the case that the cargo is attached to the N-terminus of the iRGD peptide as the disulfide bond is cleaved before the peptide is internalized black line.
Table 1: The most common peptides linear and cyclic utilized for the formulation of PDCs used in cancer. Letters with bold color stand for D-amino acids. According to the National Cancer Institute cancer. The main drawback of these original anticancer agents is their uncontrolled toxicity which results in severe side effects. Without the addition of a targeting moiety, they bear low capacity to discriminate cancerous from normal cells. Moreover, the addition of a peptide as a targeting vehicle can enhance the pharmacokinetic and therapeutic window of the parent cytotoxic agent.
Since different drugs may employ a different mechanistic approach to kill cells, the appropriate drug is selected according to features characterizing the targeted cancerous cells. For instance, daunorubicin and doxorubicin possess similar mechanisms of action  , whereas gemcitabine  , camptothecin  and paclitaxel  function through different mechanisms. Figure 4: Representative examples of anticancer drugs utilized for the construction of PDCs.
The most usual conjugation sites are marked with red cycles. The most usual c The selected drug must comply with certain design principles in order to serve as an appealing candidate for PDCs. The selected drug must be amenable to the linker chemistry. In the latter case, the site of derivatization has to be carefully selected so that the biological activity of the drug and the release of the active drug will not be perturbed. In case that the drug binds through recognition of a specific receptor, in silico approaches have to be recruited in order to rationally select the location of the drug that will be chemically modified .
Furthermore, it must be sufficiently cytotoxic versus the selected malignant tumor cells in order to eliminate them and consequently promote tumor regression. The selected drug should ideally possess low-nanomolar IC 50 values for the targeted malignant tumor. A legitimate strategy to overcome a low drug potency problem is by increasing the drug loading of the peptide-carrier.
For example, in the PDC ANG, 3 drug molecules paclitaxel were loaded on a single angiopep-2 peptide which has completed phase II clinical trials . Nevertheless, the concept of higher drug loading is hard to be implemented, in contrast with single drug loading that is usually preferred, mostly due to poor physicochemical properties. Gemcitabine Gem : Gemcitabine dFdC is a nucleoside analog of deoxycytidine in which the hydrogen atoms on the 2' carbon are replaced by fluorine. It is sold under the brand name Gemzar by Eli Lilly and Company and has been FDA approved for the treatment of various cancers including breast, ovarian, non-small cell lung and pancreatic cancer.
The main drawbacks for its use are the high and non-selective toxicity to normal cells, the deactivation through deamination in its inactive metabolite dFdU, the acquired multidrug resistance MDR and its high hydrophilicity deterring its prolonged drug release from various vehicles  , which therefore reduces the effective concentration of gemcitabine.
It enters cells through nucleoside transporters hENTs human equilibrative nucleoside transporters and hCNTs human concentrative nucleoside transporters and mostly through hENT1 human equilibrative nucleoside transporter 1 [89,90]. After internalization, gemcitabine is sequentially mono-, di- and tri-phosphorylated by phosphorylating kinases. The incorporation of dFdCTP into DNA during polymerization, which causes DNA polymerases unable to proceed, is the major mechanism by which gemcitabine causes cell death masked termination . Paclitaxel PTX : Paclitaxel PTX is a member of the taxane family and one of the most common anticancer agents used against a wide variety of tumors.
The main disadvantages in the utilization of paclitaxel are its high hydrophobicity, requiring suitable vehicles to effectively deliver it to tumor tissues, and the development of multidrug resistance due to the P-glycoprotein-mediated efflux [85,92]. Paclitaxel stabilizes microtubules by binding specifically to the beta-tubulin subunit, promoting mitotic halt and consequently cell death . The difference with other known drugs that act on microtubules vinca alkaloids is that paclitaxel does not induce the disassembly of microtubules but boosts the polymerization of tubulin .
Anthracyclines: Anthracyclines are among the main anti-cancer drugs that are applied in combinations with other chemotherapeutic agents. They are utilized against a variety of cancers including leukemias, lymphomas, breast, ovarian, bladder and lung.
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Daunorubicin Dau was the first anthracycline discovered that was extracted from Streptomyces peucetius , a species of actinobacteria, at the beginning of the s. Shortly after, the isolation of doxorubicin Dox from a mutated Streptomyces strain was accomplished. Anthracyclines are consisted of a tetracyclin aglycon part and a daunosamine sugar moiety.
The mechanism of action of anthracyclines is based on their intercalation to DNA inhibiting the macromolecular biosynthesis. They may also increase quinone type free radical production, however, this plays a role rather in their cytotoxic side effects. Daunorubicin is mainly used in the treatment of leukemia  while doxorubicin in the cure of other types of cancers breast cancer, bladder cancer, Kaposi's sarcoma in combination with other anti-cancer agents. It was first isolated and characterized in by Wall et al. This stabilized complex prevents the re-ligation step of DNA, catalyzed by topo I, resulting in DNA damage and therefore cell death apoptosis.
CPT is predominantly cytotoxic during the S phase replication of DNA because of the collision of the replication fork with the cleavable complex, converting the single-strand breaks into double-strand breaks and eventually causing cell death .
Methods in Neurosciences, Vol 13: Neuropeptide Analogs, Conjugates, and Fragments
Although CPT showed remarkable results during its phase I clinical trials against a variety of solid tumors, its low water-solubility and stability led to the formulation of various new analogs with the same mechanism of action. The two most progressed analogs of CPT are topotecan and irinotecan. Topotecan hycamtin has been approved by the FDA for the treatment of ovarian and cervical cancer, as also small cell lung carcinoma. Irinotecan camptosar has been approved by the FDA for the treatment of metastatic carcinoma of the colon or rectum, alone or in combination with fluorouracil 5-FU.
Another crucial aspect that should be considered during the design of a PDC is the linker tethering the peptide and the drug. The linker has to be carefully shaped so as not to perturb the binding affinity of the peptide to its receptor and the drug efficacy. An inappropriate linker may impede the release of the drug from the PDC and therefore diminish its overall therapeutic potency. This linker can be designed to bear an enzyme-hydrolyzable unit EHU like a carboxylic ester or an amide bond, cleaved by esterases and amidases, respectively.
The most commonly utilized linkers that bear a carboxylic ester bond, as the enzyme-hydrolyzable unit, are succinyl derived from succinic acid and glutaryl derived from glutaric acid. Also, during the design of the PDC specific attention has to be given on the selection of the bonds that will be used in the linker. Specifically, in several currently available PDCs, at least two different bonds are used: one to connect the linker to the peptide and the other to connect the drug to the linker.
Specifically, there are certain bonds like imine, oxime, hydrazone, orthoester, acetal, vinyl ether and polyketal  that are known to undergo hydrolysis at acidic pH, while being extremely stable during blood circulation. Additionally, disulfide linkers are often adopted in PDCs, since they are cleaved by reducing agents like cysteine and glutathione, present in high concentrations in malignant cells.
Linkers bearing enzyme-hydrolyzable units EHU responsive to proteases are degradable peptide linkers that have attracted significant interest due to the specificity of certain enzymes and there has been a dramatic escalation over in the past years. Para -amino benzyl alcohol PABC; colored in red is a representative example that can be connected in the amino group via an amide bond to an enzyme-hydrolyzable unit EHU; colored in green and to a tumor-targeting element i.
The EHU is designed so as to be a substrate for proteases overexpressed in the targeted tumor microenvironment i. Figure 5: Illustration of the drug release mechanism from the self-immolative spacer PABC conjugated to a tumor homing peptide via an enzyme-hydrolyzable unit. Figure 5: Illustration of the drug release mechanism from the self-immolative spacer PABC conjugated to a tum Integrating the basic design principles in PDCs pinpointed above, a list of representative developed examples is analyzed below, so as to provide a spherical perspective regarding peptide—drug conjugation chemistry.
These clinical trials will focus on chronic lymphocytic leukemia CLL. Table 3: Peptide—drug conjugates consisting of peptides and small molecules that have been used in clinical trials. Except these two PDCs, there are other types of PDCs that do not consist of peptides as targeting moieties and small molecules as drugs and have reached even up to phase III clinical trials. Table 4: Various other types of peptide—drug conjugates in clinical trials.
Notably, there is only one PDC in the market designated In-DTPA- d -Phe 1 -octreotide, which is utilized for diagnostic radiology in somatostatin receptor-positive tumors . It constitutes a complex of Indium bound to diethylenetriaminopentaacetic acid DTPA , which is conjugated to the targeting somatostatin peptide [D-Phe 1 ]-octreotide.
Recently, another similar analog, designated In-DTPA- d -Phe -1 -Asp 0 - d -Phe 1 -octreotide, has been evaluated and presented enhanced tumor accumulation in pancreatic tumor cells and simultaneously lower renal radioactivity . Specifically, Andrew V. Schally and his group first synthesized the corresponding analogs  where they covalently coupled the two drugs to the epsilon-amino group of the D-Lys side chain of the peptide D-Lys 6 -LHRH.
Notably, both conjugates fully preserved the cytotoxic activity of the parent drugs, DOX or 2-pyrrolino-DOX, respectively, in vitro and also retained the high binding affinity of their peptide carrier to receptors for LHRH on rat pituitary . The two conjugates were subjected to stability tests and they showed slow drug release in human serum in contrast with nude mice that carboxylesterase enzymes are about 10 times higher .
Consequently, the two analogs were heavily evaluated in in vivo models in nude mice bearing various types of cancer. These results were confirmed in nude mice bearing other ovarian human cancers ES-2 , where AN caused up to Shortly afterward, they tested the two conjugates in membranes of human breast cancer cells: MCF-7 hormone-dependent and MDA-MB hormone- independent .
They proved that the specific analogs retained the high binding affinity of the D-Lys 6 -LHRH carrier to the relevant receptors. AN was tested regarding the inhibition of tumor growth of subcutaneously sc implanted androgen-dependent LNCaP and MDA-PCa-2b and androgen-independent C prostate cancers, xenografted into nude mice. The results demonstrated the stronger inhibition of AN on the tumor with respect to the free DOX .
Similarly, in vivo experiments were conducted regarding AN in nude mice bearing xenografts of MDA-PCa-2b prostate cancer cells, showing identical results like AN . Treatment with DOX arrested tumor growth but did not reduce tumor volume. Based on the presented results, it can be concluded that these two analogs possess higher antitumor activity but less toxicity with respect to the parent drugs DOX and 2-pyrrolino-DOX and can be used versus a wide variety of ovarian, prostate, endometrial and breast tumors. Due to the promising results from phase II trials in endometrial cancer, a multinational phase III clinical study is underway .
It is important to note that despite the fact that analog AN presented a better biological profile, evident in all the preclinical models, its further development fell short due to chemical and plasma instability. According to ClinicalTrials. The results showed promising tolerance from the patients with fewer side effects than the commonly applied drugs. Moreover, AEZS was evaluated in phase I clinical trial on patients with castration- and taxane-resistant prostate cancer and the results proved that AEZS possesses a sufficient safety profile and efficacy.
Similarly, adding disaccharide moieties to enkephalin analogs increased their antinociceptve activity up to fold, following intravenous administration Elmagbari et al. Two important factors, namely lipophilicity and basicity, contribute to increased permeability of peptides through the blood-brain barrier without the need for specific transporters or carriers. The lipophilic character of a peptide can be altered by either conjugation to a hydrophobic moiety e. It has been shown that polyamine-modified proteins and peptides cross the blood-brain barrier more efficiently, as compared to unmodified ones Poduslo and Curran, a; b; Poduslo et al.
It has also been shown Tamai et al. In addition to direct modification of the peptides, there are a few other drug delivery strategies with improved uptake of drugs into the CNS. These include liposome-, micelle- or nanoparticle-mediated delivery of peptides through the blood-brain barrier Kreuter et al. These novel drug delivery technologies can also be applicable to neuropeptide-based compositions disclosed herein and known in the art. There are several peptide-engineering strategies to improve permeability of neuropeptides through the blood-brain barrier.
These are summarized in FIG. However, none of the studies mentioned above showed a systematic approach of combining these strategies to further boost the permeability of the peptides through the blood-brain barrier. What is disclosed herein is the application of these peptide-engineering strategies in combination to known anticonvulsant neuropeptides, such as somatostatin or galanin, thereby making these peptides anticonvulsant via the intravenous i. Disclosed herein are methods and compositions for increasing permeability through the blood-brain barrier.
For example, the rate of increase can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , , or percent when compared with the control, native, or wild type peptide or composition.
Specifically disclosed herein is a composition with increased permeability of the blood-brain barrier, wherein the composition comprises a peptide with increased lipophilic character and increased basicity when compared to the non-altered form of the peptide FIG. Also disclosed are compositions with increased permeability of the blood-brain barrier, wherein the composition comprises a peptide with increased lipophilic character, increased basicity, and increased glycosylation when compared to the non-altered form of the peptide.
Also disclosed herein are methods of increasing permeability of the blood-brain barrier for a peptide, comprising increasing lipophilic character and increasing basicity of the peptide compared to the non-altered form of the peptide. Another method of increasing permeability of the blood-brain barrier for a peptide, comprises increasing lipophilic character, increasing basicity, and increasing glycosylation of the peptide compared to a non-altered form of the peptide. The lipophilic character of the composition can be increased by conjugating the peptide to a hydrophobic moiety, for example.
Examples of hydrophobic moieties include, but are not limited to, polyaliphatic chains or aromatic residues. The lipophilic character can also be increased by increasing halogenation of aromatic residues or polyaliphatic reagents, such as perfluorohexanoic acid. The basicity of the composition can be increased by introducing homo- and heterooligomers of positively charged amino acid residues, including, but not limited to Lysine, Arginine, homo-Lysine, homo-Arginine, Ornitine in L- or D-isomer configuration; 2,3-Diaminopropioic acid; 2,4-Diaminobutyric acid. The basicity can also be increased by conjugation to polyamine-based moieties, such as spermine, spermidine, polyamidoamine dendrimers or polyamine toxins and derivatives thereof.
The glycosylation can be introduced by conjugation to xylose, glucose, galactose, maltose, maltotriose, mannose, lactose, melibiose or similar saccharides. Also disclosed are vectors comprising the compositions disclosed herein. An example of vectors able to cross the BBB can be found in Toyobuku et al. Disclosed herein are methods of treating specific diseases and disorders involving the central nervous system, or any application that involves the need for a compound to cross the blood-brain barrier.
Methods and routes of administration, dosages, and pharmaceutical compositions are discussed in more detail below. Regarding the use of the compositions disclosed herein to treat spinal cord injury and multiple sclerosis, the following references are hereby incorporated in their entirety for their teaching concerning the treatment of these diseases: Hawes J J, Narasimhaiah R, Picciotto M R Galanin and galanin-like peptide modulate neurite outgrowth via protein kinase C-mediated activation of extracellular signal-related kinase.
Eur J. Suarez V, et al, The axotomy-induced neuropeptides galanin and pituitary adenylate cyclase-activating peptide promote axonal sprouting of primary afferent and cranial motor neurons Eur J. The compositions and methods disclosed herein can also be useful in preventing anoxic damage, increasing growth hormone secretion in humans, controlling prolactin release from pituitary adenomas, prolonging morphine analgesia, as an antidepressant, and in feeding disorders, for example. Also disclosed are methods of treating pain and other neurological disorders comprising administering to a subject in need thereof an effective amount of the polypeptides disclosed herein.
The methods and compositions disclosed herein can also be used in the prevention, amelioration, or treatment of neurological disorders, such as those disclosed above and known to those of skill in the art. The methods and compositions disclosed herein can be used in conjunction with other compositions or treatment methods. The following drugs and classes of drugs can be used in combination with the compositions disclosed herein for Alzheimer disease: amyloid lowering agents, such as Flurizan; galantamine Razadyne ; rivastigmine Exelon ; donepezil Aricept ; tacrine Cognex ; memantine Namenda ; and vaccine for Alzheimer's disease.
Also disclosed are methods of treating a subject in need of a composition that crosses the blood-brain barrier, comprising identifying the composition to be used in treatment of the subject; modifying the composition by increasing lipophilicity and basicity of the composition; and administering the modified composition to the subject in need thereof. These compositions can ben. Also disclosed are methods of treating a subject in need of a composition that crosses the blood-brain barrier, comprising: identifying the composition to be used in treatment of the subject; modifying the composition by increasing lipophilicity, glycosylation, and basicity of the composition; and administering the modified composition to the subject in need thereof.
Also disclosed are methods of treating a subject in need of a composition that crosses the blood-brain barrier, comprising identifying the composition to be used in treatment of the subject; modifying the composition by increasing lipophilicity, glycosylation, and basicity of the composition; inserting the modified composition into a vector; administering the vector to the subject in need thereof.
Also disclosed is a method of treating a subject in need of a composition that crosses the blood-brain barrier, comprising: identifying the composition to be used in treatment of the subject; modifying the composition by increasing lipophilicity and basicity of the composition; inserting the modified composition into a vector; and administering the vector to the subject in need thereof. The disclosed compositions can be used in a variety of ways as research tools.
The compositions can be used, for example, as targets in combinatorial chemistry protocols or other screening protocols to isolate molecules that possess desired functional properties, such as galanin agonists or antagonists or partial agonists. The compositions can be used to discover individual and network interactions between different neuropeptides, other neurotransmitters, receptors and ion channels in the nervous system.
For example, the disclosed compositions can be used to discover synergistic interactions between galanin receptor antagonists and drugs that act on molecular targets expressed on the same neurons. The disclosed compositions can be used as discussed herein as either reagents in micro arrays or as reagents to probe or analyze existing microarrays. The disclosed compositions can be used in any known method for isolating or identifying single nucleotide polymorphisms. The compositions can also be used in any known way of using the computer readable embodiments of the disclosed compositions, for example, to study relatedness or to perform molecular modeling analysis related to the disclosed compositions.
The disclosed compositions and methods can be used for targeted gene disruption and modification in any animal that can undergo these events. For example, a gene producing galanin can be altered to express a galanin analog with increased permeability of the blood-brain barrier.
Gene modification and gene disruption refer to the methods, techniques, and compositions that surround the selective removal or alteration of a gene or stretch of chromosome in an animal, such as a mammal, in a way that propagates the modification through the germ line of the mammal. In general, a cell is transformed with a vector which is designed to homologously recombine with a region of a particular chromosome contained within the cell, as for example, described herein. This homologous recombination event can produce a chromosome which has exogenous DNA introduced, for example in frame, with the surrounding DNA.
This type of protocol allows for very specific mutations, such as point mutations, to be introduced into the genome contained within the cell. Methods for performing this type of homologous recombination are disclosed herein. One of the preferred characteristics of performing homologous recombination in mammalian cells is that the cells should be able to be cultured, because the desired recombination event occurs at a low frequency.
Once the cell is produced through the methods described herein, an animal can be produced from this cell through either stem cell technology or cloning technology. For example, if the cell into which the nucleic acid was transfected was a stem cell for the organism, then this cell, after transfection and culturing, can be used to produce an organism which will contain the gene modification or disruption in germ line cells, which can then in turn be used to produce another animal that possesses the gene modification or disruption in all of its cells.
In other methods for production of an animal containing the gene modification or disruption in all of its cells, cloning technologies can be used. These technologies generally take the nucleus of the transfected cell and either through fusion or replacement fuse the transfected nucleus with an oocyte which can then be manipulated to produce an animal. The advantage of procedures that use cloning instead of ES technology is that cells other than ES cells can be transfected.
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For example, a fibroblast cell, which is very easy to culture can be used as the cell which is transfected and has a gene modification or disruption event take place, and then cells derived from this cell can be used to clone a whole animal. Disclosed are the components to be used to prepare the disclosed compositions as well as the compositions themselves and to be used within the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc.
For example, if a particular galanin analog is disclosed and discussed and a number of modifications that can be made to a number of molecules including the variant are discussed, specifically contemplated is each and every combination and permutation of the galanin analog and the modifications that are possible unless specifically indicated to the contrary.
Likewise, any subset or combination of these is also disclosed. This concept applies to all aspects of this application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a variety of additional steps that can be performed it is understood that each of these additional steps can be performed with any specific embodiment or combination of embodiments of the disclosed methods.
Spontaneous epileptic seizures result from excessive discharge in hyperexcitable neurons primarily located in the hippocampus.
The role of these neuropeptides has been elucidated using both pharmacological and genetic knockout or overexpression approaches. For example, Oberto and coworkers Oberto et al. Similarly, Leresche and coworkers Leresche et al. Modulation of glutamate release by galanin in the hippocampus was investigated in two transgenic mouse models: knockout of galanin GalKO and overexpressing GalOE mice Mazarati et al. In GalKO and GalOE mice, depolarization-induced glutamate release was increased and decreased by centrally administered galanin, respectively, indicating a role of hippocampal galanin as an anticonvulsant through the glutamatergic system Mazarati, At least three neuropeptides and their receptors were shown to play a role in epileptogenesis: galanin, somatostatin and neuropeptide Y.
Galanin immunoreactivity in the hippocampus is diminished after limbic status epilepticus. Injection of galanin into the hippocampal dentate hilus prevented onset of limbic status epilepticus and stopped status epilepticus. It thus appears that galanin acts as an endogenous anticonvulsant that inhibits status epilepticus Mazarati et al. Evidence of this was shown by examining the phenotype of transgenic mice with overexpression of galanin Kokaia et al.
In this study, galanin suppressed kindling epileptogenesis. The role of neuropeptides in modulating neurotransmitter release and seizure control has been recognized as an opportunity for new therapeutic treatments. As described below, a number of published studies showed potent anticonvulsant activity of neuropeptides in animal models. Neuropeptide Y suppressed epileptiform activity in rat hippocampal slices in vitro Klapstein and Colmers, In another study Baraban et al.
Intracerebroventricular neuropeptide Y prevented death induced by kainic acid administration. Finally, the anticonvulsant action of neuropeptide Y was demonstrated to be mediated through the Y5 receptors Sperk and Herzog, Hippocampal opioid peptides, including dynorphin, have been implicated in epileptogenesis and epileptic seizures reviewed by Hong, and Solbrig and Koob, Seizures induced by either electroconvulsive shocks or amygdala kindling resulted in the initial release of both enkephalin and dynorphin, but also caused a long-term decrease in dynorphin Gall, Anticonvulsant activity of dynorphin was shown in the rat model of self-sustained status epilepticus Mazarati and Wasterlain, , as illustrated in FIG.
As with dynorphin, i. NPY administered into the lateral ventricle appeared to be a potent inhibitor of kainate-induced seizures Woldbye et al. It had been suggested that the antiepileptic effect was mediated by neuropeptide Y5 receptors, a finding that was confirmed in a study with Y5R-deficient mice Marsh et al. Galanin has been recognized as a potential anticonvulsant agent since the work of Mazarati and coworkers Mazarati et al.
When injected directly into the lateral brain ventricle or hippocampus, galanin decreased the severity of picrotoxin-induced kindled convulsions in rats.
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In the animal model of status epilepticus, perihilar injection of galanin, before or after perforant path stimulation PPS , shortened the duration of seizures Mazarati et al. These effects were reversed by co-application of galanin antagonists. As illustrated in FIG. One strategy for treating epilepsy is to use neuropeptide-based therapeutics. As a proof-of-concept, two non-peptide galanin receptor agonists, galnon and galmic, were recently shown to possess anticonvulsant and antiepileptic activities Saar et al.
Both compounds appeared to possess a midrange micromolar affinity for GalR1 or GalR2 receptors, and exhibited anticonvulsant activity in animal models of epilepsy when administered systemically. As shown in FIG. When injected i. Intrahippocampal injection of galnon was also demonstrated to shorten the duration of self-sustained status epilepticus.
Similarly, galmic blocked status epilepticus when injected i. Thus, these two galanin agonists are useful anticonvulsants, and validate galanin receptors as therapeutic targets for epilepsy. Galanin is a amino-acid neuropeptide, but SAR studies identified that the N-terminal portion is still a highly potent agonist as compared to the whole-length peptide Langel and Bartfai, A galanin analog can be used with the methods disclosed herein, in which the Gly 1 residue is replaced by N-methyl-Gly sarcosine, SAR , as shown below.
N-methylation of Gly 1 protected the peptide from accelerated proteolytic degradation from the N-terminus, whereas it did not significantly change its affinity for the galanin receptor Rivera Baeza et al. The same study identified that the N-terminal extensions caused a loss of the biological activity. On the other hand, the C-terminal portion of galanin appears to be very robust when it comes to attaching to larger structures Pooga et al.
Therefore, the strategy for design of [Sar 1 ]galanin analogs is similar to that used with somatostatin only with regard to amino acid replacements, but it differs by introducing the extensions at the C-, rather than at the N-terminus. Example 2. The smallest galanin analog with the most potent and long-lasting anticonvulsant activity can be obtained from GAL-BBB2.
These peptides can have increased stability when compared to galanin, for example. Each of these can also possess anticonvulsant activity. Limited structure-function relationship studies are carried out to identify the smallest fragment of the GAL-BBB2 analog that maintains anticonvulsant activity when administered systemically. Galanin analogs containing either the C-terminal and central truncations are synthesized and tested. In addition, limited structure-function relationship study of the C-terminal motif are carried out to optimize permeability of the analog through the blood-brain-barrier.
Discussed below are various galanin analogs and methods for their design and synthesis see examples 1 and 2. There are many lines of evidence showing that brain somatostatin plays an important role as an inhibitor of seizures and epileptogenesis Vezzani and Hoyer, Somatostatin is a major neuropeptide expressed in GABAergic interneurons of the hippocampus. Moreover, somatostatin release from rat hippocampal neurons was stimulated by glutamate Fontana et al.
The expression of somatostatin and its receptors is significantly changed after epileptic seizures, and this neuropeptide has also been postulated to control neuronal excitability during epileptogenesis reviewed in Schwarzer et al. Receptor-subtype-knockout and pharmacological studies have suggested the involvement of at least four subtypes of somatostatin receptors sst1, sst2, sst3 and sst4 in glutamate-mediated neurotransmission in hippocampus Pikwo et al. The recent study of Csaba and coworkers Csaba et al. The most direct evidence of the anticonvulsant activity of somatostatin comes from studying its pharmacological effects on seizures and epileptogenesis in animal epilepsy models Vezzani et al.
Injection of somatostatin or its subtype-selective analogs resulted in a reduced number of seizures, and raised the latency to seizures induced by kainic or quinolonic acid Vezzani et al. Similarly, infusion of RC, the somatostatin sst2-selective agonist decreased the number of animals with pentylenetetrazol-induced tonic-clonic seizures Perez et al.
Somatostatin is a amino-acid hypothalamic peptide with a single disulfide bridge, originally discovered in Brazeau et al. The sequence of somatostatin is shown below. The [D-Trp 8 ] or [L-Trp 8 ] somatostatin can be used as the metabolically stable analog with the methods disclosed herein. To increase basicity, Thr, Ser or Asn residues can be systematically replaced with isosterically similar, but positively charged DAB diaminobutyric acid or DAP di aminopropionic acid residues. As summarized in Table 5, nine analogs are synthesized and assayed for their affinity to somatostatin receptors.
The modifications that do not negatively affect high affinity binding are combined together. These 2 nd -generation analogs comprise combined modifications. Table 6 provides information about the structure and function of the proposed modules. Also disclosed for use with the compositions and methods disclosed herein is octreotide.
Octreotide is a somatostatin analog that more selective toward sst2 subtype of somatostatin receptors there are 5 known subtypes. Somatostatin has been shown to be involved in epilepsy and epileptogenesis. The following reference is incorporated in its entirety for its teaching concerning octreotide, somatostatin, and epilepsy: Vezzani A and Hoyer D, Eur J Neurosci, , vol 11, pp Similarly, a role of somatostatin in nociception was shown in Chapman V and Dicjkenson A H, Neuropeptides , vol 23, , herein incorporated by reference in its entirety for its teaching concerning somatostatin, octreotide, and nociception.
A role of somatostatin in a development of Alzheimer disease has recently described Saito T et al, Nature Medicine, , vol 11, p. Discussed below are methods for designing and synthesizing somatostatin analogs see Example 1. DSIP was effective in suppressing seizures in the metaphit-induced epilepsy model. Moreover, it has been shown that DSIP potentiated anticonvulsant activity of valproate in the same epilepsy model Hrncic, Stanojlovic et al. In addition, this peptide was shown to modulate interactions between enkephalins with opioid receptors, resulting in analgesic effects of DSIP Nakamura, Nakashima et al.
Neuroprotective activity of DSIP was shown in a model of toxic cerebral oedema. The compositions disclosed herein have shown increased permeability of the blood-brain barrier. As described herein, disclosed is set of neuropeptide analogs that are designed and synthesized to test their ability to bind with high affinity to their respective receptors. This set includes approximately ten analogs per neuropeptide.
High-affinity analogs are further tested for their ability to penetrate the blood-brain barrier. Results from 1st-generation analogs are followed by the synthesis and evaluation of 2nd- and, subsequently, 3rd-generation analogs. The most promising analogs are selected high-affinity ligands with enhanced permeability through the blood-brain barrier to confirm their agonist activity in functional assays.
A subset of these analogs potent agonists with enhanced permeability through the blood-brain barrier are then pharmacologically tested in vivo. To become a drug, a neuropeptide analog should possess several important features, including: 1 high potency and selectivity, 2 metabolic stability, 3 relatively long half-life and reduced clearance from systemic circulation, and 4 increased permeability through the blood-brain barrier.
Most neuropeptides exhibit high potency and selectivity. An increase in half-life and decrease in elimination rate can be efficiently achieved by conjugating a polymer-based moiety to a peptide e. Greater permeability through the blood-brain barrier can be introduced by increase in lipophilicity or cationization, as well as by adding prodrug, nutrient transport mimetic or glycosylation. The structure of an ideal drug neuropeptide is schematically shown in FIG. The cationic and lipophilic modules promote interactions with negatively charged membrane surfaces, and improve the diffusion through the membranes, respectively.
The function of the active transport mimetic structure is to increase the specificity of neuropeptide uptake into the brain by enhancing interactions with specific nutrient transporters located on the surface of the brain endothelial cells. These bulky moieties are tested as the N- or C-terminal extensions of the model neuropeptides, and more versatile positions of attachment within the neuropeptide structure are also disclosed herein.
The following strategy was used to design neuropeptide analogs with enhanced blood-brain barrier penetrability: begin with metabolically-stable analogs, if available. Identify additional AA positions in the analogs amenable to side chain replacements. Identify positions at the N- and C-termini amenable to introduction of bulky moieties.
Increase lipophilicity and basicity of analogs by side-chain replacements. Include a nutrient mimetic structure at the extension to improve specificity of the blood-brain barrier penetration. A key to the successful design of such analogs is the correct combination of the above-mentioned modifications. To achieve this goal, a systematic approach in designing and evaluating individual sets of modifications and their optimal combinations can be taken.
The general strategy is schematically illustrated in FIG. The modification of amino acids as disclosed herein can be introduced during solid-phase peptide synthesis using an automated peptide synthesizer. All non-natural amino acids or conjugated structures are as commercially available Fmoc-protected derivatives. Disclosed are analogs of galanin that have desirable properties, such as increased permeability of the blood brain barrier.
Also disclosed are analogs of somatostatin that have increased permeability of the blood-brain barrier. It is also understood that each individual analog discussed in the tables in the Examples also has a base permeability which can be determined from the disclosed activities of the composition. It is understood that these percentages of increased activity can be calculated from a base permeability of a wild type, native, or control peptide obtained at any time which provides data in the analytical range of the assay, unless otherwise indicated.
Disclosed are substitutions, deletions, modifications, additions, and extensions to the known, or wild type, peptide, as disclosed in Examples 1 and 2. For example, in Table 7, disclosed are N-terminal extensions for somatostatin. The extensions disclosed herein can be used with a native, wild type, or known peptide, or can be used in combination with an analog of a known peptide. For example, side chain modifications can be made to the known peptide, and then combined with an extension as disclosed herein. Also disclosed are amino acid substitutions and additions, wherein the substitution or addition is of a non-naturally occurring substance.
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Examples include, but are not limited to, aminovaleric or aminohexanoic acid. This can result in a minimization of the overall molecular size without significant change of spacing between key residues. The spacer can replace 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, or 50 residues, for example.
Also disclosed herein are variations of amino acids wherein their conformation has been changed. It is understood that as discussed herein the use of the terms homology and identity mean the same thing as similarity. Thus, for example, if the use of the word homology is used between two non-natural sequences it is understood that this is not necessarily indicating an evolutionary relationship between these two sequences, but rather is looking at the similarity or relatedness between their nucleic acid sequences.
Many of the methods for determining homology between two evolutionarily related molecules are routinely applied to any two or more nucleic acids or proteins for the purpose of measuring sequence similarity regardless of whether they are evolutionarily related or not. In general, it is understood that one way to define any known variants and derivatives or those that might arise, of the disclosed genes and proteins herein, is through defining the variants and derivatives in terms of homology to specific known sequences. This identity of particular sequences disclosed herein is also discussed elsewhere herein.
In general, variants of genes and proteins herein disclosed typically have at least, about 40, 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 percent homology to the stated sequence or the native sequence. Those of skill in the art readily understand how to determine the homology of two proteins or nucleic acids, such as genes. For example, the homology can be calculated after aligning the two sequences so that the homology is at its highest level.
Another way of calculating homology can be performed by published algorithms. Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. The same types of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M.
Science , , Jaeger et al. USA , , Jaeger et al. Methods Enzymol. It is understood that any of the methods typically can be used and that in certain instances the results of these various methods may differ, but the skilled artisan understands if identity is found with at least one of these methods, the sequences would be said to have the stated identity, and be disclosed herein.
For example, as used herein, a sequence recited as having a particular percent homology to another sequence refers to sequences that have the recited homology as calculated by any one or more of the calculation methods described above. For example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using the Zuker calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by any of the other calculation methods.
As another example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using both the Zuker calculation method and the Pearson and Lipman calculation method even if the first sequence does not have 80 percent homology to the second sequence as calculated by the Smith and Waterman calculation method, the Needleman and Wunsch calculation method, the Jaeger calculation methods, or any of the other calculation methods.
As yet another example, a first sequence has 80 percent homology, as defined herein, to a second sequence if the first sequence is calculated to have 80 percent homology to the second sequence using each of calculation methods although, in practice, the different calculation methods will often result in different calculated homology percentages. There are a variety of molecules disclosed herein peptides, such as various galanin and somatostatin analogs.
It is understood that these peptide based molecules can be encoded by a number of nucleic acids, including for example the nucleic acids that encode, for example, SEQ ID NOS , and it is understood that for example, when a vector is expressed in a cell, that the expressed mRNA will typically be made up of A, C, G, and U. There are a variety of sequences related to, for example, galanin, which can be found at, for example, Genbank database which can be accessed at www. These sequences and others are herein incorporated by reference in their entireties as well as for individual subsequences contained therein.
It is understood that the description related to this sequence is applicable to any sequence related to a galanin analog unless specifically indicated otherwise. Those of skill in the art understand how to resolve sequence discrepancies and differences and to adjust the compositions and methods relating to a particular sequence to other related sequences i. There are a number of compositions and methods which can be used to deliver nucleic acids or peptides to cells, either in vitro or in vivo.
The vectors disclosed herein can be used in multiple ways. In one example, the vectors disclosed herein can be used to deliver nucleic acids encoding the peptides disclosed herein to cells and subjects. Vectors can also be used with peptides to facilitate the crossing of the blood-brain barrier, as discussed above. Methods and compositions relating to vectors can largely be broken down into two classes: viral based delivery systems and non-viral based delivery systems.
For example, nucleic acids and peptides can be delivered through a number of direct delivery systems such as, electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes.
Appropriate means for transfection, including viral vectors, chemical transfectants, or physico-mechanical methods such as electroporation and direct diffusion of DNA, are described by, for example, Wolff, J. Nature, , , Such methods are well known in the art and readily adaptable for use with the compositions and methods described herein. In certain cases, the methods will be modified to specifically function with large DNA molecules. Further, these methods can be used to target certain diseases and cell populations by using the targeting characteristics of the carrier.
For the purpose of further improvement of delivering the compositions across blood-brain barrier, the TAT protein transduction domain can be used Dietz GP and Bahr M, Mol Cell Neurosci, , vol 27, p. As used herein, plasmid or viral vectors are agents that transport the disclosed nucleic acids or peptides, such as those related to galanin and somatostatin analogs, into the cell without degradation.
In some embodiments the delivery systems are derived from either a virus or a retrovirus. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviral vectors are able to carry a larger genetic payload, i. However, they are not as useful in non-proliferating cells. Adenovirus vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation, and can transfect non-dividing cells. Pox viral vectors are large and have several sites for inserting genes, they are thermostable and can be stored at room temperature.
A preferred embodiment is a viral vector which has been engineered so as to suppress the immune response of the host organism, elicited by the viral antigens. Preferred vectors of this type will carry coding regions for Interleukin 8 or Viral vectors can have higher transaction abilities than chemical or physical methods to introduce genes into cells.
Typically, viral vectors contain, nonstructural early genes, structural late genes, an RNA polymerase III transcript, inverted terminal repeats necessary for replication and encapsidation, and promoters to control the transcription and replication of the viral genome. Constructs of this type can carry up to about 8 kb of foreign genetic material. The necessary functions of the removed early genes are typically supplied by cell lines which have been engineered to express the gene products of the early genes in trans.
A retrovirus is an animal virus belonging to the virus family of Retroviridae, including any types, subfamilies, genus, or tropisms. Retroviral vectors, in general, are described by Verma, I. In Microbiology, American Society for Microbiology, pp. Examples of methods for using retroviral vectors for gene therapy are described in U.
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