Ex Parte Troy et al

Patent Trial and Appeal BoardMar 15, 2019

13768687 (P.T.A.B. Mar. 15, 2019)

UNITED STA TES p A TENT AND TRADEMARK OFFICE APPLICATION NO. FILING DATE 13/768,687 02/15/2013 21003 7590 03/19/2019 BAKER BOTTS L.L.P. 30 ROCKEFELLER PLAZA 44THFLOOR NEW YORK, NY 10112-4498 FIRST NAMED INVENTOR Carol M. Troy UNITED STATES DEPARTMENT OF COMMERCE United States Patent and Trademark Office Address: COMMISSIONER FOR PATENTS P.O. Box 1450 Alexandria, Virginia 22313-1450 www .uspto.gov ATTORNEY DOCKET NO. CONFIRMATION NO. 070050.4767 5649 EXAMINER FONTAINHAS, AURORAM ART UNIT PAPER NUMBER 1649 NOTIFICATION DATE DELIVERY MODE 03/19/2019 ELECTRONIC Please find below and/or attached an Office communication concerning this application or proceeding. The time period for reply, if any, is set in the attached communication. Notice of the Office communication was sent electronically on above-indicated "Notification Date" to the following e-mail address(es): DLNYDOCKET@BAKERBOTTS.COM PTOL-90A (Rev. 04/07) UNITED STATES PATENT AND TRADEMARK OFFICE BEFORE THE PATENT TRIAL AND APPEAL BOARD Ex parte CAROL M. TROY, NSIKAN AKPAN, GUY SALVESEN, and SCOTT SNIPES 1 Appeal2018-002370 Application 13/768,687 Technology Center 1600 Before RICHARD M. LEBOVITZ, JEFFREY N. FREDMAN, and RYAN H. FLAX, Administrative Patent Judges. FLAX, Administrative Patent Judge. DECISION ON APPEAL 1 Appellants identify the Real Parties in Interest as "The Trustees of Columbia University" and "Sanford-Burnham Medical Research Institute." Br. 3. Appeal2018-002370 Application 13/768,687 This is a decision under 35 U.S.C. Â§ 134(a) involving claims directed to a method of treating ischemic injury in the central nervous system comprising intranasally administering an effective amount of an apoptotic target inhibitor covalently conjugated to a cell-penetrating peptide to a subject in need thereof. Claims 1, 4--13, 15, 16, and 18 are on appeal as rejected under 35 U.S.C. Â§ 103 and for obviousness-type double patenting. We have jurisdiction under 35 U.S.C. Â§ 6(b). We affirm; we designate the affirmed rejections as new grounds of rejection under 35 U.S.C. Â§ 103(a), pursuant to our authority under 37 C.F.R. Â§ 4I.50(b). STATEMENT OF THE CASE Independent claim 1 is representative and is reproduced below: 1. A method of treating ischemic injury in the central nervous system comprising intranasally administering an effective amount of an apoptotic target inhibitor covalently conjugated to a cell-penetrating peptide to a subject in need thereof, where said apoptotic target inhibitor is selected from the group consisting of: isatin sulfonamides, anilinoquinazolines, nucleic acid inhibitors of caspases, antibody inhibitors of caspases, peptide inhibitors of caspases, the class of protein inhibitors identified as Inhibitors of Apoptosis ("IAPs"), and dominant negative forms of a caspase polypeptide; said cell-penetrating peptide is selected from the group consisting of penetratinl, transportan, pISl, Tat(48-60), pVEC, MAP, and MTS; and where said ischemic injury is treated thereby. Br. 14 (Claims Appendix; paragraphing added). In response to an Election of Species Requirement ( mailed Apr. 1, 2014 ), Appellants elected 2 Appeal2018-002370 Application 13/768,687 penetratinl as a cell penetrating peptide and elected caspase 6 inhibitors as an apoptotic target inhibitor. See Response to Restriction Requirement 4 (dated July 31, 2014). We limit our decision on obviousness to consideration of the elected species. The following rejections are appealed: Claims 1, 4--7, 9-11, 13, and 15 stand rejected under 35 U.S.C. Â§ 103(a) over Fan2 and Duan. 3 Final Action 3. Claims 1, 4--13, 15, 16, and 18 stand rejected under 35 U.S.C. Â§ 103(a) over Fan, Duan, and Hermel. 4 Id. at 5. Claims 1, 4--7, 9-11, 13, 15, and 16 stand rejected over claims 1, 3-5, and 8-12 of U.S. Patent Application 14/568,710 for obviousness-type double patenting. 5 Id. at 10. DISCUSSION "[T]he examiner bears the initial burden, on review of the prior art or on any other ground, of presenting aprimafacie case ofunpatentability. If that burden is met, the burden of coming forward with evidence or argument 2 Yong-Feng Fan et al., Apoptosis Inhibition in Ischemic Brain by Intraperitoneal PTD-BIR3-RJNG (XJAP), 48 NEUROCHEMISTRY INT'L 50-59 (2006) ("Fan"). 3 Xiaopin Duan & Shirui Mao, New Strategies to Improve the Intranasal Absorption of Insulin, 15 (11/12) DRUG DISCOVERY TODAY 416-27 (2010) ("Duan"). 4 E. Hermel et al., Specific Caspase Interactions and Amplification are Involved in Selective Neuronal Vulnerability in Huntington's Disease, 11 CELL DEATH & DIFFERENTIATION 424--38 (2004) ("Hermel"). 5 U.S. Patent Application 14/568,710 was abandoned Dec. 19, 2017 for failure to respond to an office action. See Notice (Dec. 22, 2017). This rejection is dismissed. 3 Appeal2018-002370 Application 13/768,687 shifts to the applicant." In re Oetiker, 977 F.2d 1443, 1445 (Fed. Cir. 1992). Arguments made by Appellants in the Appeal Brief (no Reply Brief was submitted) have been considered in this Decision; arguments not so- presented in the Brief are waived. See 37 C.F.R. Â§ 4I.37(c)(l)(iv) (2015); see also Ex parte Borden, 93 USPQ2d 1473, 1474 (BPAI 2010) (informative) ("Any bases for asserting error, whether factual or legal, that are not raised in the principal brief are waived."). As applicable to the rejections on appeal and Appellants' arguments there-over, "[t]he combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results." KSR Int'! Co. v. Teleflex Inc., 550 U.S. 398,416 (2007). Put another way, "when a patent claims a structure [or method] already known in the prior art that is altered by the mere substitution of one element for another known in the field, the combination must do more than yield a predictable result." Id. "In determining whether the subject matter of a patent claim is obvious, neither the particular motivation nor the avowed purpose of the patentee controls. What matters is the objective reach of the claim. If the claim extends to what is obvious, it is invalid underÂ§ 103." Id. at 419. "[T]he question is whether there is something in the prior art as a whole to suggest the desirability, and thus the obviousness, of making the combination, not whether there is something in the prior art as a whole to suggest that the combination is the most desirable combination available." In re Fulton, 391 F.3d 1195, 1200 (Fed. Cir. 2004) (citation omitted). "Patents [ and publications] are part of the literature of the art and are relevant for all they contain." In re Young, 927 F.2d 588, 591 (Fed. Cir. 4 Appeal2018-002370 Application 13/768,687 1991 ). That the prior art "discloses a multitude of effective combinations does not render any particular formulation less obvious. This is especially true because the claimed composition is used for the identical purpose taught by the prior art." Merck & Co. Inc. v. Biocraft Labs. Inc., 874 F.2d 804, 807 (Fed. Cir. 1989). Appellants argue the two obviousness rejections together, addressing only independent claim 1 specifically. Therefore, we address the obviousness rejections and Appellants' arguments similarly. The Examiner determined that the claims would have been obvious over the prior art combination of Fan and Duan, and in the case of dependent claims 8, 12, 16, and 18, the Examiner also added Hermel to the combination. Final Action 3-8 and Answer 2-5 (collectively citing Fan 50, 51, 52, 54, 55, 57, 58; Duan 417,421,425; and Hermel 426,428,434). The cited prior art references are directed to treating ischemic brain conditions by apoptosis inhibition and delivering drugs to the brain by pairing the active component with a penetration enhancing compound, as well as the nasal administration of such a combination. Generally, the Examiner's position is that Fan teaches and suggests an effective apoptosis inhibitor (BIR3-RING), which is fused, i.e., covalently linked, to a cell-penetrant (PTD, which is a penetratin (Pen-I)), which facilitates the transport of the therapeutic across the blood brain barrier and into the brain. See Final Action 3; Answer 3--4. Further, it is the Examiner's position that it would have been obvious to use Duan's disclosed intranasal administration method to deliver Fan's covalently conjugated therapeutic/cell-penetrant formulation because Duan teaches and suggests 5 Appeal2018-002370 Application 13/768,687 that it is an effective and convenient technique, used for a similarly conjugated insulin-CPP (cell-penetrating peptide, e.g., penetratin) formulation. See Final Action 4; Answer 4--5. We discern no error in the Examiner's determinations. We address Appellants' arguments below. Appellants argue that the combination of Duan and Fan "does not actually teach 'intranasally administering an effective amount of an apoptotic target inhibitor covalently conjugated to a cell-penetrating peptide' as recited by Claim 1," and that "the references cited by or to explain Duan" "actually teach away from covalent attachment as claimed in Claim 1, and thus also teach away from combination with Fan, which is also alleged to disclose a covalent attachment." Br. 7. Further, Appellants argue Duan does not expressly teach intranasal administration of any protein covalently attached to a CPP, even though Duan states "[ w ]hen covalently linked with a cargo, including polypeptides and oligonucleotides with many times their own molecular mass, these [cell-penetrating] peptides are still able to translocate." Br. 8 ( emphasis added). Appellants argue this does not teach any protein connected to a CPP or the nature of the bond. Id. Appellants further argue that this portion ofDuan cites Khafagy II (as reference "41"), 6 which is directed to physical mixtures of CPP and insulin, not covalently bonded components. Id. at 8-9. Appellants' argument, therefore, is that Duan does not teach or suggest covalently linking a CPP, like penetratinl, 6 El-Sayed Khafagy et al., Effect of Cell-penetrating Peptides on the Nasal Absorption of Insulin, 133 J. CONTROLLED RELEASE 103---08 (2009) ("Khafagy II"). 6 Appeal2018-002370 Application 13/768,687 and an active protein, such as insulin, or an anti-apoptotic protein, such as BIR3-RING. These arguments are not persuasive. As noted by Appellants, Duan explicitly states that CPPs (aka "protein transduction domains" or PTDs), such as penetratin, can be covalently linked with polypeptides and still translocate, i.e., transition through a membrane. Duan 421. This explicit statement in Duan is immediately followed by another statement that a conjugated insulin-Tat peptide formulation effectively transported across cell barriers. Looking to the reference cited by Duan (as reference "39") for this subsequent statement, similar to how Appellants have likewise looked to the Duan-cited Khafagy II reference, we find it directed to Liang, 7 which confirms that by "conjugated," Duan means a covalent linkage between insulin and TAT consisting of amino groups. Liang 73 5-3 6, Fig. 1 ( amino groups of insulin reacted with SMCC and then linked to TAT peptide). As argued by Appellants, Duan does cite Khafagy II (reference "41 ") in the same relevant portion. See Duan 421. However, Khafagy II is not cited there for the purpose of limiting Duan' s disclosure to only physical mixtures of CPP and insulin or as an indication that covalently linking the two was ill- 7 Jun F. Liang & Victor C. Yang, Insulin-cell Penetrating Peptide Hybrids with Improved Intestinal Absorption Efficiency, 335 BIOCHEMICAL & BIOPHYSICAL RES. COMMS. 734--38 (2005) ("Liang"). We also note that Liang discusses the background of CCPs and notes that they can be conjugated to enzymes (i.e., proteins) and cross the blood brain barrier (BBB), which also supports the Examiner's positions. See Liang 734. Liang's disclosure is a confirmation of the meaning of Duan's explicit disclosure. Because Liang was not of record and is supporting evidence as to the meaning of Duan' s disclosure, we designate our affirmance of the Examiner's rejections as new grounds of rejection. 7 Appeal2018-002370 Application 13/768,687 advised, but was cited with reference to a specific success in pairing insulin and penetratin in an intranasal administration and comparing it to a subcutaneous administration. Id. ("A dose-dependent relationship of L- penetratin and insulin bioavailability was established. The pharmacological availability and bioavailability of nasally administered insulin was up to 76.7% and 50.7% relative to the subcutaneous route, respectively [ 41]."). Therefore, as confirmed by, inter alia, Liang, Duan teaches and suggests that polypeptides, such as insulin, can be covalently linked to a CPP and that such a conjugate can be administered intranasally. Appellants' arguments that Duan is not explicit enough or indirectly teaches away are not persuasive. A "teaching away" requires a reference to actually criticize, discredit, or otherwise discourage the claimed solution. See In re Fulton, 391 F.3d 1195, 1201 (Fed. Cir. 2004). "The prior art's mere disclosure of more than one alternative does not constitute a teaching away from ... alternatives because such disclosure does not criticize, discredit, or otherwise discourage the solution claimed." In re Fulton, 391 F.3d 1195, 1201 (Fed. Cir. 2004). "Although a reference that teaches away is a significant factor to be considered in determining unobviousness, the nature of the teaching is highly relevant, and must be weighed in substance. A known or obvious composition [ or method] does not become patentable simply because it has been described as somewhat inferior to some other product for the same use." In re Gurley, 27 F.3d 551, 553 (Fed. Cir. 1994). The facts here are analogous to those upon which the Federal Circuit's decision in Gurley was based. According to our reviewing court: 8 Appeal2018-002370 Application 13/768,687 The facts in Gurley' s record are that this use of epoxy was known, the structure of these circuit boards was known, and epoxy had been used for Gurley's purpose. We share Gurley's view that a person seeking to improve the art of flexible circuit boards, on learning from Yamaguchi that epoxy was inferior to polyester-imide resins, might well be led to search beyond epoxy for improved products. However, Yamaguchi also teaches that epoxy is usable and has been used for Gurley's purpose. The Board recognized Yamaguchi' s teaching of the deficiencies of epoxy-impregnated material, but observed that Gurley did not distinguish his epoxy product from the product described by Yamaguchi. On the facts of this case, Gurley's "teaching away" argument was insufficient to establish patentability. Gurley did not off er specific epoxies, or improved properties, and we are not presented with the question of whether any such products might meet the requirements of patentability. Even reading Yamaguchi' s description as discouraging use of epoxy for this purpose, Gurley asserted no discovery beyond what was known to the art. The Board correctly held that Gurley's invention would have been obvious in view of the Yamaguchi reference in combination with the prior art set forth in Gurley's specification. Id. Here, as in Gurley, it could be said that Duan, if also considering the references it cites and all they also teach and suggest, for example Khafagy II (which is directed to the success of a physical mixture of insulin and penetratin or R8), teaches that sometimes covalently linking a therapeutic molecule and a penetrant molecule is not as good a choice as merely physically mixing these components (see also discussion infra regarding Morishita, Kamei, and Khafagy I). However, Duan teaches and suggests that such covalent linking works generally when administering a therapeutic that otherwise would experience absorption issues and would work for an intranasal administration. Therefore, it would have been obvious to the 9 Appeal2018-002370 Application 13/768,687 skilled artisan to use Duan' s delivery methods with covalently linked/fused apoptosis inhibitors and CPPs, as taught by Fan. Appellants also argue that the Morishita 8 reference, cited previously by the Examiner during prosecution (Appellants point to an Office Action dated Apr. 7, 2016), differentiates itself from the use of covalent bonds between a protein and a CPP. Br. 9. Appellants' argument is that Morishita further supports that the skilled artisan would not covalently link a CPP and a protein. This argument is not persuasive. Appellants' view of Morishita is overly narrow. Morishita is, indeed, focused on an invention where a cell- penetrating peptide is mixed with, but not covalently bonded to a drug, however, it also explicitly states that some formulations of drugs and CPPs, such as penetratin, "require [a] covalent bond between the drug [ and penetratin] to be allowed to permeate the mucosa .... " Morishita 1 :43-50, 2:43-50, 3:1-15 (citing PCT Pub. Nos. 2002/530059 and 2002/519392). Thus, Morishita does not support the position that the claimed covalently linked drug and penetrant would not have been obvious. Appellants also argue that another evidentiary reference, Kamei, 9 states that its results were based on a physical mixture of insulin and CPP, which it considered to have more potential for success and to be a more 8 See US 8,895,503 B2 (issued Nov. 25, 2014) ("Morishita"), which is the U.S. counterpart to PCT Pub. No. W02009/107766 (published Jan. 27, 2011). 9 Noriyasu Kamei et al., Permeation Characteristics of Oligoarginine Through Intestinal Epithelium and its Usefulness for Intestinal Peptide Drug Delivery, 131 J. CONTROLLED RELEASE 94--99 (2008) ("Kamei"). 10 Appeal2018-002370 Application 13/768,687 convenient strategy than covalent conjugation of the components. Br. 9-10. Appellants argue this teaches away from covalent linking of and intranasally administering proteins and CPPs. Id. at 10. This argument is also unpersuasive. Kamei's position on covalent linking can be summarized by the following: "in some case[ s] a covalent linkage between the macromolecular drug and oligoarginine may hamper the ability of oligoarginine to enhance intestinal epithelial permeation of therapeutic peptides and proteins." Kamei 99. This statement does not definitively suggest that covalent linking would not have been useful and Kamei's conclusion is specific to the facts of its study. Kamei does not teach away from the claimed invention or the prior art combination. Appellants also argue that another reference previously cited during prosecution, Khafagy I ( citing Response to Office Action dated Oct. 7, 2016), outlines a rationale for using non-covalent mixtures of protein and CPP and in the case of its method, covalently linked CPP-protein was incompatible with intranasal administration. Br. 10. This argument is not persuasive. The disclosure of Khafagy I is largely the same as or at least substantially similar to that of Khafagy II, as discussed above. The fact that Khafagy I discloses a successful nasal administration of a merely physically mixed formulation of penetratin and a protein/peptide does not suggest that covalently-linked formulations would not work or would have been disfavored. 11 Appeal2018-002370 Application 13/768,687 Appellants also discuss Li, 10 which was cited by the Examiner as disclosing covalently attaching a protein and a CPP for improved intraperitoneally administered therapy's delivery to the brain (across the blood brain barrier), arguing that Li was not a useful reference relating to intranasal administration as disclosed in Khafagy I ( and as claimed). Br. 10-11. This argument is not persuasive. The Examiner did not rely on Li for a conclusion or determination of obviousness. Therefore, whether Li establishes that results related to covalently linked proteins and CPPs administered intraperitoneally are informative about the same formulations administered intranasally is not determinative. For the reasons set forth above, we conclude that the Examiner has established that the claims would have been obvious over Fan and Duan. Appellants have not persuaded us that the Examiner erred in the obviousness rejection. Appellants do not present a separate argument over the Fan-Duan- Hermel prior art combination and respective rejection; therefore, we affirm both obviousness rejections for the same reasons, as set forth above. SUMMARY The obviousness rejections under 35 U.S.C. Â§ 103 are each affirmed. The double patenting rejection is dismissed. 10 Tianfu Li et al., In Vivo Delivery of a XIAP (BIR3-RING) Fusion Protein Containing the Protein Transduction Domain Protects Against Neuronal Death Induced by Seizures, 197 EXPERIMENTAL NEUROLOGY 301---08 (2005) ("Li"). 12 Appeal2018-002370 Application 13/768,687 TIME PERIOD FOR RESPONSE This Decision contains new grounds of rejection pursuant to 37 C.F.R. Â§ 4I.50(b), which provides, "[a] new ground of rejection pursuant to this paragraph shall not be considered final for judicial review." 37 C.F.R. Â§ 4I.50(b) also provides: When the Board enters such a non-final decision, the appellant, within two months from the date of the decision, must exercise one of the following two options with respect to the new ground of rejection to avoid termination of the appeal as to the rejected claims: ( 1) Reopen prosecution. Submit an appropriate amend- ment of the claims so rejected or new Evidence relating to the claims so rejected, or both, and have the matter reconsidered by the examiner, in which event the prosecution will be remanded to the examiner. The new ground of rejection is binding upon the examiner unless an amendment or new Evidence not previously of Record is made which, in the opinion of the examiner, overcomes the new ground of rejection designated in the decision. Should the examiner reject the claims, appellant may again appeal to the Board pursuant to this subpart. (2) Request rehearing. Request that the proceeding be reheard under Â§ 41.52 by the Board upon the same Record. The request for rehearing must address any new ground of rejection and state with particularity the points believed to have been misapprehended or overlooked in entering the new ground of rejection and also state all other grounds upon which rehearing is sought. Further guidance on responding to a new ground of rejection can be found in the Manual of Patent Examining ProcedureÂ§ 1214.01. AFFIRMED 37 C.F.R. Â§ 4I.50(b) 13 Application/Control No. Applicant(s)/Patent Under Patent Appeal No. Notice of References Cited 13/768,687 2018-002370 Examiner Art Unit 1649 Page 1 of 1 U.S. PATENT DOCUMENTS * Document Number Date Country Code-Number-Kind Code MM-YYYY Name Classification A US- B US- C US- D US- E US- F US- G US- H US- I US- J US- K US- L US- M US- FOREIGN PATENT DOCUMENTS * Document Number Date Country Code-Number-Kind Code MM-YYYY Country Name Classification N 0 p Q R s T NON-PATENT DOCUMENTS * Include as applicable: Author, Title Date, Publisher, Edition or Volume, Pertinent Pages) u Jun F. Liang & Victor C. Yang, Insulin-cell Penetrating Peptide Hybrids with Improved Intestinal Absorption Efficiency, 335 Biochemical & Biophysical Res. Comms. 734-38 (2005) V w X *A copy of this reference is not being furnished with this Office action. (See MPEP Â§ 707.05(a).) Dates in MM-YYYY format are publication dates. Classifications may be US or foreign. U.S. Patent and Trademark Office PT0-892 (Rev. 01-2001) Notice of References Cited Part of Paper No. Available online at www.sciencedirect.com SCll!NCl!@DIRl!CTÂ® BBRC ELSEVIER Biochemical and Biophysical Research Communications 335 (2005) 734--738 www.elsevier.com/locate/ybbrc Insulin-cell penetrating peptide hybrids with improved intestinal absorption efficiency Jun F. Liang a,*, Victor C. Yang b a Department of Chemistry and Chemical Biology, Stevens Institute of Technology, Hoboken. NJ 07030, USA b College of Pharmacy, University of Michigan, Ann Arbor, I1,fI 48109, USA Received 20 July 2005 Available online 3 August 2005 Abstract Cell-penetrating peptide (CPP) was linked to insulin to form insulin-CPP hybrids. The intestinal absorption efficiency of CPP hybridized insulin was 6-8 times increased compared to normal insulin as tested on Caco-2 cell monolayer, a widely used in vitro model for intestinal absorption. Insulin-CPP hybrid transportation seemed to be through an active and transcytosis-like mecha- nism. Importantly, insulin in hybrids kept intact after they passed through the Caco-2 cell monolayer. This study provides a new clue for oral insulin development. Â© 2005 Elsevier Inc. All rights reserved. Keywords: Cell-penetrating peptide; Diabetes; Oral insulin; Caco-2 cell monolayer; TAT; Drug delivery The most physiological way of insulin administration is oral mode because on adsorption in intestines, insulin unambiguously lands in portal system, more closely approximating what occurs in the non-diabetic individ- ual. Parenteral insulin treatment frequently produces a peripheral hyperinsulinemia which has been linked to hypertension and development of atherosclerosis [1]. In addition, current dosage regimens of insulin comprise of up to four subcutaneous injections per day in order to minimize the long-term complications of diabetes melli- tus, [2]. Compliance with such demanding dosing regi- mens is difficult, making the development of an oral form clearly appealing. As an oral dosage form providing adequate bioavail- ability of insulin would certainly revolutionize the treat- ment of diabetes, numerous attempts have so far been undertaken in order to reach this goal [3-8]. The efficacy of oral delivery systems for insulin, however, is strongly * Corresponding author. Fax: -+-I 201 216 8240. E-mail address: jliang2@stevens.edu (J.F. Liang). 0006-291X/$ - see front matter Â© 2005 Elsevier Inc. All rights reserved. doi: 10.1016/j .bbrc.2005.07 .142 limited by the rapid insulin degradation because of the presence of intestinal proteases and the low insulin absorption rate due to epithelial cell layer on gastroin- testinal mucosa. Although enzyme degradation can be overcome through the co-injection of insulin with cer- tain enzyme inhibitors, the low insulin absorption rate is always a barrier for oral insulin. Insulin must traverse at least two cell membranes (inner and outer cell mem- brane on the two sides of epithelial cell layer) to enter the circulation. Recently, a group of peptide named cell-penetrating peptide (CPP) has been discovered and extensively stud- ied. CPPs can translocate across almost all types of plas- ma membrane of eukaryotic cells through a seemingly energy-independent pathway [9,10]. In addition, by chemically hybridizing with other macromolecules or even nanoparticles, CPPs can translocate all these linked specifies into cells or tissues. It has been demonstrated that CPPs can even bring conjugated enzymes to cross blood-brain barrier (BBB) [11 ], a tight junction formed by endothelial cells, to delivery enzyme into the brain. The objective of this research is to examine if CPPs JF. Liang, V C. Yang I Biochemical and Biophysical Research Communications 335 (2005) 734-738 735 can help linked insulin cross the epithelial cell layer on gastrointestinal mucosa and improve the poor absorp- tion rate of oral insulin. Materials and methods Materials. Porcine FITC-labeled insulin (insulin/FITC 'Â°"1:1) was purchased from Sigma. Caco-2 cells were obtained from American Type Culture Collection (Manassas, VA). Twenty-four wells transwell culture plates were from Corning (Corning, N'Y). All other chemicals used were commercial products of reagent grade. CPP TAT (CGGGYGRKKRRORRR) was synthesized by Protein Core Facility at University of Michigan. The underlined sequence of TAT peptide is the core sequence of the peptide that is responsible for the membrane translocation function of TAT peptide. Another four amino acid res- idues were added at the N-terminal of the peptide in order to introduce SH group (cysteine) for the conjugation to insulin and enhance the flexibility of the peptide (GGG sequence). Synthesis of insulin/TAT conjugates. FITC-labeled insulin (F-insu- lin) was used in the whole experiment. F-insulin was dissolved in phosphate-buffered saline (PBS, 3.0 ml, pH 8.2) at a concentration of 2.0 mg/ml. After addition of 20 Âµl triethylamine (TEA), 200 ~11 succ- inimidyl 4-( N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC, 1.5 mg/ml) was added. The mixture was incubated at room tempera- ture for 2 h. Free SMCC and TEA were removed from F-insulin through the ultrafiltration using 1.0 K molecular weight cut mem- brane. After adjusting the pH of the F-insulin solution to 5.5, TAT peptide (1.0mg TAT in 0.2ml PBS) was added. The mixture was incubated at room temperature for 2 hand then kept at 4 Â°C overnight. F-insulin/TAT conjugates were separated from free F-insulin and TAT by passing the mixtures through a heparin column with a gradient elution containing 2.0 M NaCl solution [12]. Salts in collect~ F-in- sulin/TAT conjugate fraction were removed by passing the sample through a desalt column washing â€¢,vith HBSS. Successful conjugation ofF-insulin/TAT conjugates was confirmed by SDS-PAGE and Mass spectrometry. In vitro intestinal absorption assay on cultured Caco-2 cell mono- layer. The cultivation of Caco-2 cells was performed as described previously. In the transcytosis assay, F-insulin and F-insulin/TAT conjugate (2.0 Âµg/ml in HBSS) were added into either the apical (1.0 ml) or the basal (2.0 ml) chambers. Cells were incubated at 37 Â°C and samples from either apical or basal media were collected at various time intervals. Concentrations of F-insulin or F-insulin/T AT in these samples were measured using a fluorescence spectrometer with the excitation wavelength at 488 nm and emission wavelength at 510 nm. The effective permeability (Peff) was calculated based on the appear- ance of drug in the receiver chamber under sink conditions: Perr=~:= (d~~--;R) (dJ, where dCR/dt is the change in drug concentration in the receiver cham- ber at steady-state, V R is the volume of receiver bufter. A is the cross- sectional area of the exposed tissue, and CD is the drug concentration in the donor chamber. Insulin cell uptake assay using fluorescence microscopy Caco-2 cells were grmvn on chamber slides (Nalge Nunc. Naperville, USA). Cultured monolayers of Caco-2 cells were rinsed three times with HBSS (pH 6.0) and then incubated with F-insulin or F-insulin/TAT for 30 min. After removal of the culture medium, cells were washed with HBSS for three times and followed by cold acidic solution (28mM CH3COONa, I20mM NaCl, and 20mM barbital-HCI, pH 3.0) to remove all cell surface-bound F-insulin and F-insulin/TAT. After that, cells were washed with PBS and fixed with PBS-buffered (pH 7) 4% formaldehyde/1.5% methanol solution at 4 Â°C for 15 min. The coverslips were put on slides coated with buffered mounting medium consisting of90% glycerol/IO% PBS with 0.1% NaN3 and 3% DABCO to prevent fading. Examination was done with inverted Zeiss laser scanning microscope (LSM410: Carl Zeiss, Jena, Germany). Maximum excitation was performed by a 488-nm line of internal Ar- gon laser, and fluorescence emission was observed above 515 nm with long-pass barrier filter LP-515. Insulin integrity assay. A dual concentration method was used to determine the integrity of insulin. F-insulin/TAT conjugate was loaded to the top chamber of Caco-2 cell seeded transwells. After incubation for 2 h, sample was collected from the receiver of transwell and diluted into a series of solutions. Insulin concentrations in these diluted solutions were determined by both ELISA and fluorescence spec- trometry assay. ELISA was designed to measure the intact F-insulin while fluorescence spectrometry assay was designed to measure both the intact F-insulin and insulin degradation products with FITC labels. The correlation between obtained two sets of insulin concentration data was a linear plot. Intact and enzyme digested F-insulin/TAT were used as negative and positive controls. respectively. For ELISA, polystyrene plates were coated (37 Â°C, 30 min) with monoclonal anti- insulin antibodies at a concentr~tion of 5 Âµg/ml. After incubation with solution collected from the receiver of transwell at 37 Â°C for 1 0 h monoclonal anti-insulin IgG, followed by anti-mouse IgG conjug~ted with peroxidase (1:1000 dilutions). was applied to the plate and incu- bated at 37 Â°C for another 1.0 h. The bound enzyme activity was developed with 0-phenylendiamine in a citrate buffer, pH 5.0, con- taining 0.015Â°/., H 20 2 . The optical density was read at 492 nm with a microplate reader. For fluorescence spectrometry assay, F-insulin concentrations were examined by fluorescence spectrometer with the excitation wavelength at 488 nm and emission wavelength at 510 run. Results and discussion Synthesis of insulin/TAT conjugates The amino groups of insulin reacted with SMCC and then linked to a well-studied CPP, TAT peptide, through the thiol group of cysteine residue in the synthe- sized TAT peptide. The chemical linkage between insu- lin and TAT peptide was demonstrated in Fig. lA. Since both insulin and TAT peptide were linked to cross-link- er SMCC through covalent bonds, insulin/TAT conju- gates were stable and could not disassociate in absorption experiments. In addition, the CGGG se- quence between insulin and the functional sequence (underlined) of TAT peptide ensured the maximal main- tenance of TAT peptide's cell-penetrating activity. Successful synthesis ofF-insulin/TAT conjugates was primarily confirmed by SDS-PAGE assay. In addition to the insulin band, a new band at high molecular weight region was found after the reaction (Fig. lB, left). Since the high molecular weight band was visible in the fluo- rescence imaging of SDS-PAGE {Fig. 1B, middle), it might represent synthesized F-insulin/TAT conjugates. Reaction mixture was subjected to a further purification on affinity HPLC. Free insulin did show any heparin binding ability and thus was eluted out at the beginning of the elution. Insulin/TAT conjugate had moderate heparin affinity due to the presence of positively charged TAT peptide and thus was eluted out from the column a little bit earlier than free TAT peptide (Fig. 1 C). Purified 736 J.F. Liang, V C. Yang I Biochemical and Biophysical Research Communications 335 (2005) 734-738 A LysB29 0 ~ . â€¢ ___/\__,.. ;r-s-Cys-GGGYGKKRRQRRR Insulin- NH-c~ ..... H2 - N II / 0 0 B Fig. 1. Synthesis of insulin-penetrating peptide conjugates. (A) Covalent linkage of TAT peptide to insulin through cross-linker SMCC; (B) SDS- PAGE assay of insulin-FITC/TAT conjugate. SDS-PAGE gel was either stained with Commassie blue (left) or exposed to Lv illustration (middle and right) and the pictures were taken by Polaroid camera. Insulin-FITC/TAT samples were from reaction mixture (left and middle) and collected insulin-FITC/T AT fraction of HPLC (right). Lane 1, insulin-FITC; lane 2, insulin-FITC/TAT conjugate. (C) Elution profile of insulin-FITC and TAT reaction mixture through an affinity HPLC. Column: Heptrp column (1.0 ml). Elution solutions: (A) 20 mM Tris buffer solution (pH 7.2); (BJ 20 mM Tris+ 2.0 M NaCl solution (pH 7.2). Flow rate: 1.0 ml/min. insulin/TAT conjugate showed a single band on SDS- PAGE (Fig. lB, right). There are three amino groups (Gly,\.1, Phem, and LysB29) in each insulin molecule. However, since the pKa's of these amino groups are reported as 8.4, 7.1, and 9.8, respectively, insulin can be selectively labeled by adjusting reaction solution's pH value. It has been demonstrated that, under a basic condition (pH 9.5), only the amino group of LysB29 can react with an elec- trophilic reagent and produced LysB29-linked insulin conjugates [13,14]. In this experiment, the addition of TEA brought the final pH of conjugation solution to about 9.5, and thus TAT peptide should be selectively linked to the amino group of LysB29 but not amino groups of GlyA1 and PheB 1 in insulin. The LysB29 link- age of TAT peptide in synthesized insulin/TAT conju- gate was confirmed by MALDI-TOF mass spectroscopy assay. The final insulin/TAT conjugate had a molecular weight of 7704/7702 (insulin, 5807 / 5805; TAT, 1678), implying that insulin/TAT ratio in insulin/TAT conjugate was 1:1 (Fig. lA). Intestinal absorption assay using in vitro Caco-2 cell mono/ayer model Intestinal absorption ofF-insulin and F-insulin/TAT conjugate was compared in vitro on transwell-grown Caco-2 cell monolayer, a widely used in vitro model for intestinal absorption study. Caco-2 cells, which are derived from a human colorectal carcinoma, form a highly polarized membrane when grown to confluence on microporous filters and show close morphological and functional similarities with intestinal epithelium [15]. The transporting efficiency of F-insulin/T AT conjugate was tested for both basolateral-to-apical and apical-to-basolateral flux. As shown in Fig. 2A, although the transportation efficacy of insulin crossing Caco-2 cell monolayer was extremely low (Fig. 2AJ, it was dramatically increased once insulin was linked to CPP. The transporting efficiency of insulin/TAT conju- gate was 5-8 times higher than that of free insulin as demonstrated by calculated effective permeability (Petr) (Table 1) from insulin and insulin/TAT conjugate. It should be noted that unlike free insulin, no significant effective permeability between basolateral-to-apical and apical-to-basolateral flux had been found for insulin/ TAT conjugate (3.62-E6 vs 2.36-E6 for insulin and l.72-E5 vs l.62-E5 for insulin/TAT), suggesting that insulin/TAT conjugate crossing Caco-2 monolayer should be through an active mechanism. This result is in agreement with the finding that TAT peptide-medi- ated cell translocation is through an energy-independent and active pathway [9,10]. To further clarify the translocation mechanism of insulin/TAT conjugate, cell uptake and intracellular dis- tribution patterns of insulin/TAT conjugate were exam- ined on cultured Caco-2 cell monolayer using fluorescence microscopy. As shown in Fig. 2B, F-insu- lin/T AT-treated cells were associated with much high fluorescence intensity compared to F-insulin-treated cells. This result excluded the possibility that F-insu- lin/T AT passing Caco-2 cell mono layer through the junction formed by Caco-2 cells and suggested that entering into Caco-2 cells was the first step for insulin/ TAT conjugate transportation. It was interesting that although free TAT had been reported to locate in the nuclei in almost all in vitro studies [16,17], F-insulin/ TAT conjugate showed different intracellular distribu- JF. Liang, V C. Yang I Biochemical and Biophysical Research Communications 335 (2005) 734-738 737 A 0.30 0.6 0.25 1 0.20 :::1. Apical-to-Basolateral ,-_ 0.5 e Cl) 0.4 :i Basolateral-to-Apical <.) <.) = 0.15 0 <.) = 0 0.3 <.) .5 = 0.10 CL) = ----.A.- Ins ----e-Ins-TAT ;Â§ = er. 0.2 = -a-Ins -.-Ins-TAT .... .... 0.05 0.1 0.00 0 2 4 6 8 10 10 Incubation time (hr) Incubation time (hr) B Fig. 2. In vitro intestinal absorbance of insulin-penetrating peptide conjugates. (A) Apical-to-basolateral and basolateral-to-apical penneabilities of insulin-FITC (0.45 ftM) and insulin-FITC/TAT conjugate (0.45 ÂµM) across transwell-grown Caco-2 cell monolayer. Samples were collected at the indicated time points and insulin concentrations were determined by fluorescence spectrometer. Each data point represents average results from three wells; (Bl cell uptakes of insulin-FITC and insulin-FITC/TAT conjugate. Caco-2 cells were exposed to 0.5 ÂµM insulin-FITC or 0.5 ÂµM insulin-FITC/ TAT conjugate at 37 Â°C for 30 min. Insulin-FITC ( a) or insulin-FITC/TAT (b) treated cells were subjected to acid wash before fixation. Maximum excitation was performed by a 488-nm line of internal Argon laser, and fluorescence emission was observed above 515 nm with long-pass barrier filter LP-515. Table I Comparison of the effective permeability of insulin and insulin/TAT conjugate Apical-to-basolateral Basolateral-to-apical Insulin Insulin/TAT Insulin Insulin/TAT Petr (cm/s) 2.36 X 10-6 1.62 x 10-5 3.62 X 10-6 1.72 X 10-5 tion patterns (Fig. 2B). F-insulin/TAT conjugate tended to locate in cytoplasm rather than in the nuclei. There- fore, insulin/TAT conjugate crossing Caco-2 cell mono- layer seemed to be through a transcytosis-like mechanism: insulin/TAT conjugate got into Caco-2 cells from one side and got out from the other side of the cells. In fact, such "transcytosis" activity has been dem- onstrated before for CPPs including TAT peptide. It has proven that TAT is able to cross a tight junction formed by endothelial cells to bring conjugated enzymes into the brain and kidney [11 ]. Integrity assay of the insulin passing through the Caco-2 cell mono/ayer Since insulin/TAT conjugate transportation was through a transcytosis-like mechanism, insulin degrada- tion inside Caco-2 cells became interesting. Intracellular degradation of insulin would low the intestinal absorp- tion rate of insulin/TAT conjugate and become a chal- lenge for CPP based oral insulin concept. A dual concentration method was used to determine the integri- ty of insulin after they passed through the Caco-2 cell monolayer. Insulin concentrations were determined by both ELISA and fluorescence spectrometry assay. Fluo- rescence spectrometry results reflected a sum of intact F- insulin and FITC containing F-insulin degradation products while ELISA results represented only the intact insulin in the same sample. Samples were collected from the receivers of transwells loaded with insulin/TAT con- jugate. Intact insulin/TAT and enzyme-degraded insu- lin/TAT were used as negative and positive controls, respectively. As shown in Fig. 3, a linear plot was ob- tained for each sample using two sets of data obtained from fluorescence and ELISAs (Fig. 3). Plots from two collected samples and intact insulin/TAT overlapped and all these three plots had almost the same slopes. On the contrary, plot from enzyme-degraded insulin/ TAT conjugate had small slope value and was distinct from other plots from both intact insulin/TAT conju- gate and collected samples. This result suggested that most of insulin was intact after they passed through 738 J.F. Liang, V C. Yang I Biochemical and Biophysical Research Communications 335 (2005) 734-738 20-.------------~-----------, 18 16 oil 14 = '-:' 12 Â§ 10 u ~ 8 :J 6 ~ 4 2 -Integrity --sample-1 ---o-- Sample-2 - Degradation O+-....,"""c...--~~~-~~-~~-~~-~~--.------1 0 5 10 15 20 25 30 35 Fluorescence Cone. (ng) Fig. 3. Insulin integrity assay for transcytosis insulin/TAT conjugate. Insulin concentration in series of diluted samples were measured by both ELISA and fluorescence spectrometer assay. Results from ELISA were plotted against the results obtained from fluorescence spectrom- etry assay. Intact (integrity) and enzyme-degraded (degradation) insulin/TAT conjugates were used as negative and positive control, respectively. The integrity of insulin in two samples (sample l and sample 2) collected from the receiver of transwells was tested. Caco-2 cell monolayer. Therefore, although intracellular degradation of insulin might exist, it could not be a problem for CPP-mediated insulin transportation and intestinal absorption. Taken together, our research shows the CPP can im- prove the intestinal absorbance of insulin. 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