Indian Patents. 214374:PEGYLATED T20 POLYPEPTIDE COMPOUND

Title of Invention	PEGYLATED T20 POLYPEPTIDE COMPOUND
Abstract	Pegylated T20 polypeptide compounds are provided. Also provided are pharmaceutical compositions containing pegylated T20 polypeptide compounds, and processes of making and using such compounds and compositions.

Full Text	Pegylated T20 polypeptide The present invention relates to pegylated T20 polypeptide compounds, and to related methods of using and making such compounds, such as in pharmaceutical compositions and therapeutic methods of treatment. Some viruses, especially HIV, must undergo a complex process called fusion in order to enter the host cell and reproduce. DuR1ng fusion, the outer membrane of the virus fuses with the membrane of the host cell. In the case of HIV, the outer membrane of the HIV virus fuses with the membrane of the CD4+ T cell duR1ng reproduction. T20 is a member of a new class of antiviral agents that inhibit virus/membrane fusion. In the case of HIV, this provides two salutary effects: the reproduction of HIV is blocked and resultant death of the CD4+ T cells does not occur. Data from two large, internationally conducted Phase 111 tR1als indicate that combination therapy with T-20 reduced HIV to undetectable levels in the blood in at least twice the percentage of patients and provided an improved immune response at 24 weeks, as compared to those who took combination therapy without T-20. Additionally, those receiving T-20 were less likely to expeR1ence hierological failure or relapse over 24 weeks. In the first Phase 111 tR1al, conducted in North AmeR1ca and Brazil, 37 percent of patients who were treated with T-20 in combination with an optimized background regimen had undetectable blood levels (less than 400 copies/mL) of HIV at 24 weeks, compared to 16 percent who received an optimized background regimen alone (p The pR1mary efficacy endpoint for the study, the mean difference in the magnitude of decrease in HIV between the two groups in the study, was 0.934 logic copies/mL (p Results from the second Phase 111 clinical tR1al, conducted in Europe and Australia, were consistent with findings from the first study, 28 percent of patients who were treated with T-20 in combination with an optimized background regimen had undetectable blood levels (less than 400 copies/mL) of HIV at 24 weeks, compared to 14 percent receiving an optimized background regimen alone (P The mean difference in the magnitude of decrease in HIV between the two arms at 24 weeks was 0.78 log10 copies/mL (p At entry, an optimized background regimen (consisting of three to five drugs, including up to two newly approved or investigational drugs, if appropR1ate) was chosen for each patient based on treatment history and antiretroviral resistance testing. After selection of the regimen, patients were randomized 2:1 to receive either the regimen in combination with T-20 or the regimen alone. Patients randomized to T-20 received T-20 administered as one 90 mg subcutaneous self-injection twice-daily. Viral resistance to currently approved anti-HlV drugs is a significant issue in the clinical management of HIV today. Many patients who begin combination anti retroviral treatment with currently approved medications will develop resistance to one or more of these agents over time. Research suggests, however, that T20 may be unaffected by resistance to any of the currently approved antiretroviral! classes. (Data presented at the 5th International Workshop on Drug Resistance and Treatment Strategies in Scottsdale, AR1zona, June 4-8, 2001). Additional expeR1ments show that the in vitro activity of T20 is not affected by mutations associated with resistance to reverse transcR1ptase inhibitors and protease inhibitors. Like many polypeptide therapeutic agents, T20 is generally administered by injection. Current therapeutic protocols often involve more than one daily injection. It would, therefore, be advantageous to provide T20 polypeptides and pharmaceutical compositions having improved performance and pharmacokinetic characteR1stics. It would be particularly advantageous to provide for lower therapeutic doses of T20, less frequent administrations, and/or extended duration of action. These and other objects of the present invention are descR1bed in greater detail below. The present invention provides a compound of formula: wherein R1 is a capping group, m is from 1 to 17, n is from 10 to 1,000, p is from 1 to 3, and NHT20 is a T20 polypeptide covalently bonded through its terminal a-amino group. In one embodiment of the compound of the present invention R1 is ethoxy, m is 1, n is from 100 to 750, and p is 3. Also provided is a pharmaceutical composition compR1sing, in admixture with a pharmaceutically acceptable recipient, a compound of formula (I), wherein R1, m, n, p, and NHT20 are defined as above. - ^ ^ In one embodiment of the pharmaceutical composition of the present invention R1 is ethoxy, m is 1, n is from 100 to 750, and p is 3. The present invention further provides a method of inhibiting HIV infection compR1sing administeR1ng a pharmaceutical composition compR1sing, in admixture with a pharmaceutically acceptable recipient, a compound of formula (I), wherein R1, m, n, p, and NHT20 are defined as above. In one embodiment of the method of inhibiting HIV infection R1 is methoxy, m is 1, n is from 100 to 750, and p is 3. If the present invention relates to a "method for making" a "process for making" is meant. Further provided is a method for making a pegylated T20 polypeptide compR1sing reacting a T20 polypeptide with a polyethylene glycol aldehydes of formula: wherein R-i, m, n, n, and p are defined as above to produce a compound of formula (I): wherein the polyethylene glycol aldehyde molecule is bonded to the N-terminal amino group of the T20 polypeptide. If the present invention relates to a "method of inhibiting HIV infection compR1sing a compound", a "use of a compound for the preparation of a medicament of the inhibition of HIV" is meant. Also provided by the invention is a compound of formula: CH3-O-(CH2-CH2-O)n-CH2-CH2-O-CH2-CH2-CH2-NHT20 (111) wherein n is from 10 to 1,000 and NHT20 is a T20 polypeptide covalently bonded through its terminal a-amino group. Also provided by the invention is a pharmaceutical composition compR1sing, in admixture with a pharmaceutically acceptable excipient, a compound of formula (111), wherein n is from 10 to 1,000 and NHT20 is a T20 polypeptide covalently bonded through its terminal a-amino group. Also provided is a method of inhibiting HIV infection compR1sing administeR1ng a pharmaceutical composition compR1sing, in admixture with a pharmaceutically acceptable excipient, a compound of formula (III), wherein n is from 10 to 1,000 and NHT20 is a T20 polypeptide covalently bonded through its terminal a-amino group. As noted above, T20 is a "fusion inhibitor" polypeptide. T20 consists of 36 amino acids. The polypeptide sequence of T20 is: YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF[SEQ.ID.N0:1] The N-terminus (or amino terminus) amino acid is tyrosine (Y). The C-terminus (or carboxyl terminus) amino acid is phenyalanine (F). As descR1bed in Figure 1 of U.S. Patent No. 5,464,933 (SEQ ID:1), which is hereby incorporated by reference in its entirety, the T20 polypeptide sequence may be blocked/deR1vatized at one or both of its amino and carboxy termini. As descR1bed in U.S. Patent No. 5,464,933, the tyrosine amino terminus may be bocked/deR1vatized with an acryl group and the phenylalanine carboxy-terminus may be blocked/deR1vatized with an amino group (the latter resulting in a conversion of the -COOH -> -CONH2). As used herein, "T20" shall be understood to mean [SEQ.ID.NO:1], optionally blocked at the phenylalanine C-terminus with an amino group. In other words, when reference is made to "T20," the phenylalanine C-terminus is either -COOH or CONH2. The present invention provides pegylated T20 compounds of the following formula: 0 II R1(CH2CH20)n-CH2CH2-0-(CH2)m-C-NH-(CH2)p-CH2-NHT20 (I) wherein R1 is a capping group, m is from 1 to 17, n is from 10 to 1,000, p is from 1 to 3, and NHT20 is a T20 polypeptide covalently bonded through its terminal a-amino group. As used herein the R1 "capping group" is any suitable chemical group which, depending upon preference, is generally unreactlve or generally reactive with other chemical moieties. In the above compound the polyethylene glycol is covalently bonded to the a-amino group of T20. The R1 capping group is selected to permit or prevent bifunctionality, e.g., covalent attachment to a second chemical moiety of interest. In the case that the capping group is generally uncreative with other chemical moieties, R1 is relatively inert and therefore will not covalently bond with another chemical moiety. Suitable generally unreactive R1 capping groups include: hydrogen, hydroxyl, lower alky!, lower alkoxy, lower cycioalkyI, lower alkenes, lower cycloalkenyl, aryl, and heteroaryl. As used herein, the term "lower alkyl", means a substituted or unsubstantiated, straight-chain or branched-chain alkyl group containing from 1 to 7, preferably from 1 to 4, carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec.butyl, tert.butyi, n-pentyl, n-hexyl, n-heptyl and the like. The term "lower alkoxy" means a lower alkyl group as defined earlier which is bonded via an oxygen atom, with examples of lower alkoxy groups being methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec.butoxy, tert.butoxy, n-pentoxy and the like. The term "lower cycloalkyi" means a substituted or unsubstituted cycloalkyi group containing from 3 to 7, preferably from 4 to 6, carbon atoms, i.e. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl. As used herein, the term "lower alkenyl" means a substituted or unsubstituted, straight-chain or branched-chain alkenyl group containing from 2 to 7, preferably from 2 to 5, carbon atoms, e.g., ethenyl, butenyl, pentenyl, hexenyi and the like. The term "lower cycloalkenyl" means a substituted or unsubstituted, cycloalkenyl group containing from 4 to 7 carbon atoms, e.g., cyclobutenyl, cyclopentenyl, cyclohexenyl and the like. The term "aryl" means a phenyl or naphthyl group which is unsubstituted or optionally mono- or multiply-substituted by halogen, lower alkyl, lower alkoxy, tR1fluoromethyl, hydroxy!, carboxylic acid, carboxylic ester, nitro, amino, or phenyl, particularly by halogen, lower alkyl, lower alkoxy, tR1fluoromethyl, hydroxyl, nitro, amino and phenyl. The term "heteroaryl" means a 5- or 6-membered heteroaromatic group which contains one or more hetero atoms selected from N, S, and O and which may be benz-fused and/or substituted in the same manner as "aryl" defined earlier. Preferred generally unreactive R1 capping groups include methoxy, hydroxyl, or benzyloxy. -An especially preferred,-R1 capping group is methoxy. When R1 is methoxy the pegylated polypeptide compounds are sometimes referred to herein, in part, as "mPEG" compounds, wherein the "m" stands for methoxy. If the R1 capping group is generally reactive with other chemical moieties, then R1 is a functional group capable of reacting with some functional group, such as an amine and/or sulfhydryl in a peptide and/or protein. In such a case, R1 may be a functionai group that is capable of reacting readily with electrophilic or nucleophilic groups on other molecules, in contrast to those groups that require strong catalysts or highly impractical reaction conditions in order to react. If R1 is relatively reactive, the polyethylene glycol aldehyde may covalently bond with another chemical moiety. Examples of suitable generally reactive R1 capping groups include: halogen, epoxide, maleimide, orthopyR1dyl disulfide, tosylate, isocyanate, hydrazine hydrate, cyanuR1c halide, N-succinimidyloxy, sulfo-N-succinimidyloxy, 1-benzotR1azolyloxy, 1-imidazolyloxy, p-nitropheny!oxy, and O II -CH2CH2-0-(CH2)m-C-NH-(CH2)p-CH0. The term "halogen" means fluoR1ne, chloR1ne, bromine, or iodine. A preferred generally reactive R1 capping group is O • II -CH2CH2-0-(CH2)ni-C-NH-(CH2)p-CHO. When this R1 capping group is present, it will be appreciated that in the compounds of the present invention the first m, n, and/or p may be the same or different from the second m, n, and/or p in the formula. It is preferred, however, that both m"s have the same value, both n"s have the same value, and both the p"s have the same value. In the present invention, m is from 1 to 17. In a preferred embodiment, m is from 1 to 14. More preferably m is from 1 to 7, and even more preferably, m is from 1 to 4. Most preferably, m is 1. In the present invention, n is from 10 to 1,000. In a preferred embodiment of the present invention n is from 20 to 1,000. Preferably, n is from 50 to 1,000, even more preferably n is from 75 to 1,000. Most preferably, n is from 100 to 750. In the present invention, p is from 1 to 3. Preferably, p is 3. In preferred embodiments, p is 3, R1 is methoxy, m is 1, and n is from 100 to 750; or p is 2, R1 is methoxy, m is 1, and n is from 100 to 750; or p is 1, R1 is methoxy, m is 1, and n is from 100 to 750. The present invention provides embodiments of formula (I), wherein R1 is methoxy, m is 1, n is from 100 to 750, p is 3, and NHT20 is a T20 polypeptide covalently bonded through its terminal a-amino group. As noted above, the pegylated T20 compounds of the invention covalently link the a-amino group of T20 to a polyethylene glycol deR1vative having a particular structure. The pegylated compounds may be made in any manner desired, but generally they are prepared by reacting T20 with separately prepared polyethylene glycol deR1vatives. The T20 polypeptide may be prepared in any suitable manner. For example, the compounds may be synthesized using the classic MerR1field solid phase synthesis techniques involving a solid phase method employing Boc-amino acid (Chem. See, 85, 2149, 1963), by using manual or automated procedures, using a solid pliase method employing an Fmoc-amino acid ( Sheppard, R.C. et al., J.Chem. Soc. Chem. Comm., pp. 165-166 (1985)), using an Advanced Chemtech mode! 200 available from Advanced Chemtech., Louisville, Ky., using a Millipore 9050+ available from Millipore, Bedford Mass, or other available instrumentation. T20 may be produced by incorporating cDNA coding compounds of the invention into functional viral or circular plasmid DNA vectors. The vectors or plasmids can be used to transfect or transform selected microorganisms. The transformed or transected microorganisms can be cultured under conditions that are conducive to express vector-borne DNA sequences and isolation of the desired peptides from the growth medium can be achieved. (See, for example United States Patent No. 5,955,422, the entirety of which is incorporated herein by reference as if recited in full.) T20 may also be prepared by standard recombinant DNA technology using techniques that are well known in the art. For example, the procedures outlined in Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd edition, (Cold SpR1ng Harbor Press, Cold SpR1ng Harbor, N.Y.) or Ausubel et a!., Current Protocols in Molecular Biology . John Wiley and Sons, New York (1995), both of which are herein incorporated by reference. A particular method for making T20 is descR1bed in U.S. Patent, 6,015,881 which is hereby incorporated. After cleavage and deportation, T20 may be puR1fied by any suitable means. For example, ion exchange, gel filtration chromatography and/or a reverse-phase column/HPLC system can be used to puR1fy full length T20 from fragments thereof. In the case when a T20 precursor is first prepared, with a blocking/protecting group attached to the N-terminus (e.g., acyl group) and/or the C-terminus {e.g., amino group), one or both of those groups may be removed using known techniques. The amino acid sequence of T20 may be confirmed and identified using standard amino acid analysis as well as manual and automated Edman degradation and determination of each amino acid. HPLC analysis and mass spectrometry may also be used to veR1fy the production of T20. Polyethylene glycol aldehyde compounds which may be reacted with T20 nnay also be made in any desired manner. It is preferred, however, that the polyethylene glycol be made in accordance with the methods descR1bed in concurrently filed U.S. Patent Application SeR1al Number 60/398,196 filed July 24, 2O02 entitled "Polyethylene Glycol Aldehydes," the entirety of which is hereby incorporated by reference. Generally, a polyethylene glycol aldehyde of the formula: wherein R1, m, n, and p are defined as above is used to pegylate the T20. The polyethylene glycol aldehyde used to pegylated the T20 may be prepared by any suitable means. One preferred polyethylene glycol aldehyde is prepared as follows: Polyethylene glycol aldehydes of varying size {e.g., varying n values) may be prepared by following the general reaction scheme above. The pegylated T20 compounds of the present invention may be prepared by any suitable means. Further provided by the invention, however, is a method for pegylating a T20 polypeptide compR1sing reacting a T20 polypeptide, NHT20, with a polyethylene glycol aldehyde of formula: wherein R1, m, n, n, and p are defined as above; to produce a compound of formula: wherein the polyethylene glycol aldehyde molecule is bonded to the N-terminal amino group of the T20 polypeptide. The pegylated T20 is prepared by adding T20 and the PEG reagent in a molar ratio range of 1:1 to 1:100. The T20 has a free a-amino group (any acyl group is removed) and either a free carboxy group or an amino-protected carboxy group, as discussed above. The reaction mixture is placed in a borate, phosphate, or tR1 buffer at room temperature or 4 degrees Celsius for about .5 to 24 hours at a pH range of 5.5 to 7.4. The molar ratio of PEG reagent to peptide/proteins is between 1:1 to 100:1. The concentration of peptide/proteins is between 1 to 10 mg/ml. The concentration of buffer is usually 10 to 500 mM. The pegylated T20 is puR1fied by taking the reaction mixture of pegylated T20 " and diluting it with an equilibration buffer (20mM TR1s, pH 7.5). The resulting mixture is then applied on a Q-Sepharose column. After the mixture is applied on the QA column, it is washed with the equilibration buffer eluted with 75 M NaCl; eluted with 200 mM NaCI; eluted with 1M NaCl; and regenerated with 1M HOAC + 1M NaCl and 0.5 NaOH. By using reverse phase HPLC, a it is possible to readily separate and isolate the N-terminal, monopegylated product from other byproducts in the mixture. In preferred embodiments of the pegylated T20 polypeptides of the present invention, p is 3, R1 is methyl, m is 1, and n is from 100 to 750; or p is 2, R1 is methoxy, m is 1, and n is from 100 to 750; or p is 1, R1 is methoxy, m is 1, and n is from 100 to 750. The present invention also provides a pegylated T20 polypeptide of the following formula: wherein n is from 10 to 1,000 and NHT20 is a T20 polypeptide covalently bonded through its terminal a-amino group. In one embodiment n is approximately 225, for example 227. This pegylated T20 polypeptide may be made in any desired manner, preferably, it is made by the method descR1bed in Example 3. The pharmaceutical compositions of the invention comprise, in admixture with a pharmaceutically acceptable excipient, a compound of formula (1), wherein R1, m, n, p, and NHT20 are defined as above. The pharmaceutical compositions of the present invention comprising pegylated T20 polypeptides or the salts thereof, may be manufactured in any desired manner, e.g., by means of conventional mixing, encapsulating, dissolving, granulating, emulsifying, entrapping, or lyophilizing processes. These pharmaceutical preparations may be formulated with therapeutically inert, inorganic or organic excipients and carriers. Suitable excipients for injection include water, alcohols, polyols, glycerine, vegetable oils, phospholipids and surfactants. The pharmaceutical preparations may also contain preserving agents, solubilizing agents, stabilizing agents, wetting agents, emulsifying agents, sweetening agents, coloring agents, flavoring agents, salts for varying the osmotic pressure, buffers, coating agents, or antioxidants. They may also contain other therapeutically valuable substances, including additional active ingredients. The formulations suitable for parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intratracheal, and epidural) administration may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the pegylated T20 polypeptides and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the pegylated T20 polypeptides with liquid carriers. Formulations suitable for parenteral administration include: aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) conditions requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use. Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as herein above recited, or an appropriate fraction thereof, of the administered ingredient. Preferably, the pegylated T20 polypeptide is in unit dosage form. As used herein, "unit dosage form," means that an amount appropriate for a single dose the pegylated T20 polypeptide is in a premature and/or prepackaged form. This allows for convenient preparation of the pegylated T20 polypeptide for administration, and may even allow for self-administration by the patient. The unit dosage amount will obviously depend on the amount of pegylated T20 polypeptide to be delivered, and the frequency of dosing. The pegylated T20 polypeptide may also be provided in a lyophilized powder form in a unit dosage amount, suitable for reconstitution with a pharmaceutically acceptable excipient just prior to the time of administration. A particular pharmaceutical composition of the invention comprises, in admixture with a pharmaceutically acceptable excipient, a compound of formula (I), wherein R1 is methoxy, m is 1, n is from 100 to 750, and p is 3. Another pharmaceutical composition of the invention is a pharmaceutical composition comprising, in admixture with a pharmaceutically acceptable excipient, a compound of formula (III), wherein n is from 10 to 1,000 and NHT20 is a T20 polypeptide covalently bonded through its terminal a-amino group. In one embodiment n is approximately 225, for example 227. The present invention further provides methods of inhibiting HIV infection comprising administering to a patient a pharmaceutical composition comprising, in admixture with a pharmaceutically acceptable excipient, a compound of formula (I), wherein Ri, m, n, p, and NHT20 are defined as above. The pegylated T20 polypeptides are generally administered in the manner (unlegislated) T20 polypeptides are presently administered. Modifications may be made, however, to take advantage of the improved pharmacokinetic properties of the pegylated T20 polypeptides. In the method of inhibiting HIV of the invention, the pharmaceutical composition may be administered in any suitable manner and route. In a preferred method the pegylated T20 polypeptide is administered in the form of an injectable solution or suspension. Preferably, the injectable solution or suspension is administered by subcutaneous injection or intravenously. In another preferred method, the pegylated T20 polypeptide is administered though a transdermal delivery device, e.g., a transdermal patch. In the method of inhibiting HIV of the invention, the pharmaceutical composition may be administered in any suitable dosage and schedule. The pharmaceutical compositions of the invention can be administered in any form, and via any route, desired. Generally, however, the pegylated T20 polypeptides of the present invention are administered parent rally, for example, in the form of injection solutions. Determination of a therapeutically effective amount is within the skill in the art, and the therapeutically effective amount or dosage of a pegylated T20 polypeptide according to this invention may vary and will be adjusted to the individual requirements in each particular case. In general, in the case of parenteral administration to adult humans weighing approximately 70 Kg, a daily dosage of about 5 mg to about 300 mg, preferably from about 50 mg to about 200 mg, should be appropriate, although the upper limit may be exceeded when indicated. The dosage may be administered as a single dose, in divided doses, or as continuous infusion. Daily and, more preferably, weekly administrations may be employed. The present invention also provides a method of inhibiting HIV infection comprising administering a pharmaceutical composition comprising, in admixture with a pharmaceutically acceptable excipient, a compound of formula (I), wherein R1 is methoxy, m is 1, n is from 100 to 750, and p is 3. Also contemplated within the scope of the invention is a method of inhibiting HIV infection comprising administering a pharmaceutical composition comprising, in admixture with a pharmaceutically acceptable excipient, a compound of formula (111), wherein n is from 10 to 1,000 and NHT20 is a T20 polypeptide covalently bonded through its terminal a-amino group. In one embodiment n is approximately 225, for example 227. The following examples are provided to further illustrate the compounds, compositions, and methods of the present invention. These examples are illustrative only and are not intended to limit the scope of the invention in any way. Example 1 Preparation of PEG10K-butanoaldehyde mPEG of molecular weight 10,000 (30.0 g, 3 mmol) in 240 mL of toluene was azeotropically dried by refluxing for 2 hours, followed by the removal of 120 mL of toluene. The resulting solution was cooled to room temperature then potassium tert-butoxide (0.68 g, 6 mmol) in 20 ml of absolute tert-butanol and 20 ml of toluene was added to the PEG solution. The resulting mixture was stirred for two hours at room temperature under argon. Tert-butyl bromoacetate (1.00 mL, 6.75 mmol) was added to the reaction via syringe and the reaction was stirred overnight at room temperature under argon. The reaction solution was then condensed by rotary evaporation. The residue was precipitated by addition to diethyl ether. The precipitated mPEGiok t-butyl carboxymethyl ester product was filtered off and dried in vacuo. Yield: 28 g. NMR (de-DMSO): 1.40 ppm (t, 9H, -CH3); 3.21 ppm (s, - mPEG10k t-butyl carboxymethyl ester (26.5 g) was then dissolved in 350 mL of 1N sodium hydroxide and the solution was stirred at room temperature overnight. The pH of the mixture was adjusted to 2.5 by addition of 6 N hydrochloric acid, and the mixture was extracted with dichloromethane. The organic layer was dried over sodium sulfate, filtered, concentrated, and precipitated into diethyl ether. The product m-PEG10k-carboxymethyl acid was collected by filtration and dried under vacuum. Yield: 24 g. NMR (de-DMSO): 3.21 ppm (s, -OGH3); 3.5 ppm (s, -O-CH2CH2-O-); 3.99 ppm (s, 2H, -O-CH2-COOH). mPEGiok-carboxymethyl acid (6 g, 0.6 mmol) was then dissolved in anhydrous dichloromethane (30 mL) followed by the addition of 4-aminobutylraldehyde diethylacetal (140 ml, 0.9 mmol), 1-hydroxybenzotriazole (80 mg, 0.6 mmol), and dicyclohexylcarbodiimide (160 mg, 0.78 mmol). The mixture was stirred overnight at room temperature under argon. The reaction mixture was filtered, concentrated, and precipitated with mixture of 2-propanol and diethyl ether (1:1). The mPEGiok-butanoacetal product was dried in vacuo overnight. Yield: 5.4 g. NMR (de-DMSO): 1.07-1.12 ppm (t, 6H, (-0-CH2-CH3)2); 1-46 ppm (m, 4H, -NHCH2CH2CH2-CH-); 3.08-3.11 ppm (q, 2H, -NHCH2CH2CH2-CH-); 3.21 ppm (s, -OCH3); 3.5 ppm (s, -O-CH2CH2-O-); 3.85 ppm (s, 2H, -O-CH2-CO-NH-); 4.44 ppm (t, 1H, -NHCH2CH2CH2-CH-); 7.67 ppm (-NH-). mPEG10k-butanoacetal (2 g, 0.2 mmo!) was then dissolved in 20 mL of 80 % CF3COOH and the solution was stirred at room temperature overnight. The pH of the mixture was adjusted to 6.0 by addition of 1 N NaOH solution, and sodium chloride (10 wt %) was added and then the pH of the solution was adjusted to 7.0 by addition of 1 N NaOH. The mixture was extracted with dichloromethane. The organic layer was dried over sodium sulfate, filtered, concentrated, and precipitated into diethyl ether. The product mPEGiok-butanoaldehyde was collected by filtration and dried under vacuum. Yield: 1.7 g. NMR (ds-DMSO): 3.21 ppm (s, -OCH3); 3.5 ppm (s, -O-CH2CH2-O-); 3.85 ppm (s, 2H, -O-CH2-CO-NH-); 7.67 ppm (-NH-); 9.66 ppm (-CHO-). Example 2 Peqvlation of T20 with PEGioK-butanoaldehyde Butanoaldehyde of PEG lOkDa (from Example 1) was added to 15 mg of T20 (purity 93.7 %) in 3.0 ml of buffer (50 mM potassium phosphate pH 6.5) in a molar ratio of 5 moles of reagent per one mole of T20. The T20 polypeptide was deacylated at the a-amino terminus, but protected at the carboxyl terminus by -NH2. To the reaction mixture 10% (V/V) of 0.5 M sodium cyanoborohydride solution in water was added and stirred for 4 hours at room temperature. Pegylated T20 was purified from the reaction mixture using ion exchange chromatography (QA). A linear gradient with increasing salt concentrations from 150 mM to 1 M NaC! in 20 mM Tris, pH 7.5 was used to separate pegylated T20 and unmodified T20. Example 3 Peqylation of T20 with mPEG10k-propionaldehyde ; A propionaldehyde of PEG lOkDa, having the following structure is used. 150 mg of mPEGIOk-propionaldehyde was added to 15 mg of T20 (purity 93.7 %) in 3.0 ml of buffer (50 mM potassium phosphate pH 6.5) in a molar ratio of 5 moles of reagent per one mole of T20. The T1249 polypeptide was deacylated at the a-amino terminus, but protected at the carboxyl terminus by -NH2. To the reaction mixture 10% (V/V) of 0.5 M sodium cyanoborohydride solution in water was added and stirred for 4 hours at room temperature. Pegylated T20 was purified from the reaction mixture using ion exchange chromatography (QA). The structure of the pegylated T20 polypeptide follows; A linear gradient with increasing salt concentrations from 150 mM to 1 M NaCl in 20 mM Tris, pH 7.5 was used to separate pegylated T20 and unmodified T20. Example 4 Inhibitor Concentration for Pegylated T20 Phenotypic susceptibility is usually quantified in terms of IC50 or IC90 a measure of the concentration of drug needed to inhibit 50% or 90%, respectively, of viral growth. Inhibitor concentration 50 and inhibitor concentration 90 results for the pegylated T20 with PEG10K-propionaldehyde (Example 3) and pegylated T20 with mPEGIOk-butanoaldehyde (Example 2) has been reproduced in Table 1 below: Example 5 MAGI/MAG Antiviral Assays These assays score for reduction of infectious virus titer employing tine indicator cell lines MAG! (Multinuclear Activation of a Galactosidase lndicator)or the CCRS-expressing derivative cMAGI. The MAGI cell line was derived from parental HeLa cells by introducing genes for CD4 and an HlV-1 LTR-driven b-gal reporter with an amphotropic retrovirus vector (Kimpton J, Emerman M, J Virol 66:2232-9, 1992). The cMAGI cell line was derived from the MAGI cell line by introduction of the OCRS gene using the amphotropic retroviral vector, PA317 (Chackerian B, Long EM, Luciw PA, Overbaugh J, J Virol 71:3932-9, 1997.). The cMAGI cells support replication of primary NSI (R5) isolates and laboratory adapted X4 viruses, while the MAGI cells support replication of only X4 viruses. Both cell lines exploit the ability of HIV-1 tat to transactivate the expression of a b-galactosidase reporter gene driven by the HIV-LTR. The b-gal reporter has been modified to localize in the nucleus and can be detected with the X-gal substrate as intense nuclear staining within a few days of infection. The number of stained nuclei can thus be interpreted as equal to the number of infectious virions in the challenge inoculum if there is only one round of infection prior to staining. An inhibitor of infection and cell-cell fusion,e.g., T20 or T1249 (Wild C, Greenwell T, Matthews T, AIDS Res Hum Retroviruses 9:1051-3, 1993), was added 24 hrs post-infection in order to permit a readout that confidently represents a single round of infection. Infected cells were enumerated using a CCD-imager and both primary and laboratory adapted isolates showed a linear relationship between virus input and the number of infected cells visualized by the imager. In the MAGI and cMAGl assays a 50% reduction in infectious titer (VnA/o = 0.5) is significant and provides the primary cutoff value for assessing antiviral activity. A 90% reduction in infectious titer (VNNo) is used as an additional cutoff value on assessing antiviral activity. Each test compound dilution was tested in duplicate against a virus inoculum adjusted to yield approximately 1500-2000 infected cells/well of a 48 well micro titer plate. The test compound was added to the cMAGI or MAGI cells, followed by the virus inocula, and 24 hrs later, an inhibitor of infection and cell-cell fusion (Wild C, Greenwell T, Matthews T, AIDS Res Hum Retroviruses 9:1051-3, 1993) was added to prevent secondary rounds of infection and cell-cell virus spread, The cells were cultured for 2 more days, fixed and stained with the X-gal substrate to detect infected cells. The number of infected cells for each control and test compound dilution were determined with the CCD-imager. IC50 is defined as the dilution of a text compound resulting in a 50% reduction in infectious virus titer. IC90 is defined as the dilution resulting in a 90% reduction in infectious titer. The invention being thus described, it will be obvious that the same may be r varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention and all such modifications are intended to be included within the scope of the following claims. WE CLAIM : A compound of formula (I): wherein Ri is a capping group, mis from 1 to 17, n is from 10 to 1,000, pis from 1 to 3, and NHT20 is a T20 polypeptide covalently bonded through its terminal a-amino group. 2. A compound according to claim 1, wherein Ri is selected from the group consisting of halogen, epoxide, maleimide, orthopyridyl disulfide, tosylate, isocyanate, hydrazine hydrate, cyanuric halide, N-succinimidy!oxy, sulfo-N- succinimidyloxy, 1-benzotriazolyloxy, 1-(midazolyloxy, p-nitrophenyloxy, and 3, A compound according to claim 1, wherein p is 3. 4. A compound according to claim 1, wiierein p is 3, R1 is methoxy, m is 1, and n is from 100 to 750. 5. A compound of formula: wherein n is from 10 to 1,000 and NHT20 is a T20 polypeptide covalently bonded tlirough its terminal a-amino group. 6. A pharmaceutical composition comprising, in admixture with a pharmaceutically acceptable excipient, a compound of formula: wherein R1 is a capping group, m is from 1 to 17, n is from 10 to 1,000, p is from 1 to 3, and NHT20 is a T20 polypeptide covalently bonded through its terminal a-amino group. 7. A pharmaceutical composition according to claim 6, wherein p is 3, R1 is methoxy, m is 1, and n is from 100 to 750. 8. A pharmaceutical composition according to claim 6 in the form of a lypholized powder. 9. A pharmaceutical composition according to claim 6 in the form of an injectable solution or suspension, 10. A process for attaching a polyethylene glycol molecule to a T20 polypeptide comprising reacting a T20 polypeptide with a poiyethylene giyco! aldehyde of formula: wherein R is a capping group, m is from 1 to 17, n is from 10 to 1,000, and p is from 1 to 3; to produce a compound of formula: R1(CH2CH2O)n-CH2CH2-O-(CH2)m-CO-NH-(CH2)p-CH2-NHT20 (1) wherein the polyethylene glycol aldehyde molecule is bonded to the N-terminai amino group of the T20 polypeptide.

Full Text

Pegylated T20 polypeptide
The present invention relates to pegylated T20 polypeptide compounds, and to related methods of using and making such compounds, such as in pharmaceutical compositions and therapeutic methods of treatment.
Some viruses, especially HIV, must undergo a complex process called fusion in order to enter the host cell and reproduce. DuR1ng fusion, the outer membrane of the virus fuses with the membrane of the host cell. In the case of HIV, the outer membrane of the HIV virus fuses with the membrane of the CD4+ T cell duR1ng reproduction.
T20 is a member of a new class of antiviral agents that inhibit virus/membrane fusion. In the case of HIV, this provides two salutary effects: the reproduction of HIV is blocked and resultant death of the CD4+ T cells does not occur.
Data from two large, internationally conducted Phase 111 tR1als indicate that combination therapy with T-20 reduced HIV to undetectable levels in the blood in at least twice the percentage of patients and provided an improved immune response at 24 weeks, as compared to those who took combination therapy without T-20. Additionally, those receiving T-20 were less likely to expeR1ence hierological failure or relapse over 24 weeks.

In the first Phase 111 tR1al, conducted in North AmeR1ca and Brazil, 37 percent of patients who were treated with T-20 in combination with an optimized background regimen had undetectable blood levels (less than 400 copies/mL) of HIV at 24 weeks, compared to 16 percent who received an optimized background regimen alone (p The pR1mary efficacy endpoint for the study, the mean difference in the magnitude of decrease in HIV between the two groups in the study, was 0.934 logic copies/mL (p Results from the second Phase 111 clinical tR1al, conducted in Europe and Australia, were consistent with findings from the first study, 28 percent of patients who were treated with T-20 in combination with an optimized background regimen had undetectable blood levels (less than 400 copies/mL) of HIV at 24 weeks, compared to 14 percent receiving an optimized background regimen alone

(P The mean difference in the magnitude of decrease in HIV between the two arms at 24 weeks was 0.78 log10 copies/mL (p At entry, an optimized background regimen (consisting of three to five drugs, including up to two newly approved or investigational drugs, if appropR1ate) was chosen for each patient based on treatment history and antiretroviral resistance testing. After selection of the regimen, patients were randomized 2:1 to receive either the regimen in combination with T-20 or the regimen alone. Patients randomized to T-20 received T-20 administered as one 90 mg subcutaneous self-injection twice-daily.
Viral resistance to currently approved anti-HlV drugs is a significant issue in the clinical management of HIV today. Many patients who begin combination anti retroviral treatment with currently approved medications will develop resistance

to one or more of these agents over time. Research suggests, however, that T20 may be unaffected by resistance to any of the currently approved antiretroviral! classes. (Data presented at the 5th International Workshop on Drug Resistance and Treatment Strategies in Scottsdale, AR1zona, June 4-8, 2001). Additional expeR1ments show that the in vitro activity of T20 is not affected by mutations associated with resistance to reverse transcR1ptase inhibitors and protease inhibitors.
Like many polypeptide therapeutic agents, T20 is generally administered by injection. Current therapeutic protocols often involve more than one daily injection.
It would, therefore, be advantageous to provide T20 polypeptides and pharmaceutical compositions having improved performance and pharmacokinetic characteR1stics. It would be particularly advantageous to provide for lower therapeutic doses of T20, less frequent administrations, and/or extended duration of action.
These and other objects of the present invention are descR1bed in greater detail below.

The present invention provides a compound of formula:

wherein
R1 is a capping group,
m is from 1 to 17,
n is from 10 to 1,000,
p is from 1 to 3, and
NHT20 is a T20 polypeptide covalently bonded through its terminal a-amino group.
In one embodiment of the compound of the present invention R1 is ethoxy, m is 1, n is from 100 to 750, and p is 3.
Also provided is a pharmaceutical composition compR1sing, in admixture with a pharmaceutically acceptable recipient, a compound of formula (I),
wherein R1, m, n, p, and NHT20 are defined as above. - ^ ^
In one embodiment of the pharmaceutical composition of the present invention R1 is ethoxy, m is 1, n is from 100 to 750, and p is 3.

The present invention further provides a method of inhibiting HIV infection compR1sing administeR1ng a pharmaceutical composition compR1sing, in admixture with a pharmaceutically acceptable recipient, a compound of formula (I), wherein R1, m, n, p, and NHT20 are defined as above.
In one embodiment of the method of inhibiting HIV infection R1 is methoxy, m is 1, n is from 100 to 750, and p is 3.
If the present invention relates to a "method for making" a "process for
making" is meant.
Further provided is a method for making a pegylated T20 polypeptide compR1sing reacting a T20 polypeptide with a polyethylene glycol aldehydes of formula:

wherein R-i, m, n, n, and p are defined as above
to produce a compound of formula (I):
wherein the polyethylene glycol aldehyde molecule is bonded to the N-terminal
amino group of the T20 polypeptide.
If the present invention relates to a "method of inhibiting HIV infection compR1sing a compound", a "use of a compound for the preparation of a medicament of the inhibition of HIV" is meant.
Also provided by the invention is a compound of formula:
CH3-O-(CH2-CH2-O)n-CH2-CH2-O-CH2-CH2-CH2-NHT20 (111) wherein n is from 10 to 1,000 and NHT20 is a T20 polypeptide covalently bonded through its terminal a-amino group.

Also provided by the invention is a pharmaceutical composition compR1sing, in admixture with a pharmaceutically acceptable excipient, a compound of formula (111),
wherein n is from 10 to 1,000 and NHT20 is a T20 polypeptide covalently bonded through its terminal a-amino group.
Also provided is a method of inhibiting HIV infection compR1sing administeR1ng a pharmaceutical composition compR1sing, in admixture with a pharmaceutically acceptable excipient, a compound of formula (III),
wherein n is from 10 to 1,000 and NHT20 is a T20 polypeptide covalently bonded through its terminal a-amino group.
As noted above, T20 is a "fusion inhibitor" polypeptide. T20 consists of 36 amino acids. The polypeptide sequence of T20 is:
YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF[SEQ.ID.N0:1]
The N-terminus (or amino terminus) amino acid is tyrosine (Y). The C-terminus (or carboxyl terminus) amino acid is phenyalanine (F).
As descR1bed in Figure 1 of U.S. Patent No. 5,464,933 (SEQ ID:1), which is hereby incorporated by reference in its entirety, the T20 polypeptide sequence may

be blocked/deR1vatized at one or both of its amino and carboxy termini. As descR1bed in U.S. Patent No. 5,464,933, the tyrosine amino terminus may be bocked/deR1vatized with an acryl group and the phenylalanine carboxy-terminus may be blocked/deR1vatized with an amino group (the latter resulting in a conversion
of the -COOH -> -CONH2).
As used herein, "T20" shall be understood to mean [SEQ.ID.NO:1], optionally blocked at the phenylalanine C-terminus with an amino group. In other words, when reference is made to "T20," the phenylalanine C-terminus is either -COOH or CONH2.
The present invention provides pegylated T20 compounds of the following formula:
0
II R1(CH2CH20)n-CH2CH2-0-(CH2)m-C-NH-(CH2)p-CH2-NHT20 (I)
wherein
R1 is a capping group,
m is from 1 to 17,
n is from 10 to 1,000,
p is from 1 to 3, and
NHT20 is a T20 polypeptide covalently bonded through its terminal a-amino group.

As used herein the R1 "capping group" is any suitable chemical group which, depending upon preference, is generally unreactlve or generally reactive with other chemical moieties. In the above compound the polyethylene glycol is covalently bonded to the a-amino group of T20. The R1 capping group is selected to permit or prevent bifunctionality, e.g., covalent attachment to a second chemical moiety of interest.
In the case that the capping group is generally uncreative with other chemical moieties, R1 is relatively inert and therefore will not covalently bond with another chemical moiety. Suitable generally unreactive R1 capping groups include: hydrogen, hydroxyl, lower alky!, lower alkoxy, lower cycioalkyI, lower alkenes, lower cycloalkenyl, aryl, and heteroaryl.
As used herein, the term "lower alkyl", means a substituted or unsubstantiated, straight-chain or branched-chain alkyl group containing from 1 to 7, preferably from 1 to 4, carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec.butyl, tert.butyi, n-pentyl, n-hexyl, n-heptyl and the like.
The term "lower alkoxy" means a lower alkyl group as defined earlier which is bonded via an oxygen atom, with examples of lower alkoxy groups being methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec.butoxy, tert.butoxy, n-pentoxy and the like.
The term "lower cycloalkyi" means a substituted or unsubstituted cycloalkyi group containing from 3 to 7, preferably from 4 to 6, carbon atoms, i.e. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl.

As used herein, the term "lower alkenyl" means a substituted or unsubstituted, straight-chain or branched-chain alkenyl group containing from 2 to 7, preferably from 2 to 5, carbon atoms, e.g., ethenyl, butenyl, pentenyl, hexenyi and the like.
The term "lower cycloalkenyl" means a substituted or unsubstituted, cycloalkenyl group containing from 4 to 7 carbon atoms, e.g., cyclobutenyl, cyclopentenyl, cyclohexenyl and the like.
The term "aryl" means a phenyl or naphthyl group which is unsubstituted or optionally mono- or multiply-substituted by halogen, lower alkyl, lower alkoxy, tR1fluoromethyl, hydroxy!, carboxylic acid, carboxylic ester, nitro, amino, or phenyl, particularly by halogen, lower alkyl, lower alkoxy, tR1fluoromethyl, hydroxyl, nitro, amino and phenyl.
The term "heteroaryl" means a 5- or 6-membered heteroaromatic group which contains one or more hetero atoms selected from N, S, and O and which may be benz-fused and/or substituted in the same manner as "aryl" defined earlier.
Preferred generally unreactive R1 capping groups include methoxy, hydroxyl, or benzyloxy. -An especially preferred,-R1 capping group is methoxy. When R1 is methoxy the pegylated polypeptide compounds are sometimes referred to herein, in part, as "mPEG" compounds, wherein the "m" stands for methoxy.
If the R1 capping group is generally reactive with other chemical moieties, then R1 is a functional group capable of reacting with some functional group, such

as an amine and/or sulfhydryl in a peptide and/or protein. In such a case, R1 may be a functionai group that is capable of reacting readily with electrophilic or nucleophilic groups on other molecules, in contrast to those groups that require strong catalysts or highly impractical reaction conditions in order to react. If R1 is relatively reactive, the polyethylene glycol aldehyde may covalently bond with another chemical moiety.
Examples of suitable generally reactive R1 capping groups include: halogen, epoxide, maleimide, orthopyR1dyl disulfide, tosylate, isocyanate, hydrazine hydrate, cyanuR1c halide, N-succinimidyloxy, sulfo-N-succinimidyloxy, 1-benzotR1azolyloxy, 1-imidazolyloxy, p-nitropheny!oxy, and
O
II -CH2CH2-0-(CH2)m-C-NH-(CH2)p-CH0.
The term "halogen" means fluoR1ne, chloR1ne, bromine, or iodine. A
preferred generally reactive R1 capping group is
O •
II -CH2CH2-0-(CH2)ni-C-NH-(CH2)p-CHO. When this R1 capping group is present, it
will be appreciated that in the compounds of the present invention the first m, n,
and/or p may be the same or different from the second m, n, and/or p in the
formula. It is preferred, however, that both m"s have the same value, both n"s have
the same value, and both the p"s have the same value.

In the present invention, m is from 1 to 17. In a preferred embodiment, m is from 1 to 14. More preferably m is from 1 to 7, and even more preferably, m is from 1 to 4. Most preferably, m is 1.
In the present invention, n is from 10 to 1,000. In a preferred embodiment of the present invention n is from 20 to 1,000. Preferably, n is from 50 to 1,000, even more preferably n is from 75 to 1,000. Most preferably, n is from 100 to 750.
In the present invention, p is from 1 to 3. Preferably, p is 3.
In preferred embodiments, p is 3, R1 is methoxy, m is 1, and n is from 100 to 750; or p is 2, R1 is methoxy, m is 1, and n is from 100 to 750; or p is 1, R1 is methoxy, m is 1, and n is from 100 to 750.
The present invention provides embodiments of formula (I),
wherein R1 is methoxy, m is 1, n is from 100 to 750, p is 3, and NHT20 is a T20 polypeptide covalently bonded through its terminal a-amino group.
As noted above, the pegylated T20 compounds of the invention covalently link the a-amino group of T20 to a polyethylene glycol deR1vative having a particular structure. The pegylated compounds may be made in any manner desired, but generally they are prepared by reacting T20 with separately prepared polyethylene glycol deR1vatives.
The T20 polypeptide may be prepared in any suitable manner. For example, the compounds may be synthesized using the classic MerR1field solid

phase synthesis techniques involving a solid phase method employing Boc-amino acid (Chem. See, 85, 2149, 1963), by using manual or automated procedures, using a solid pliase method employing an Fmoc-amino acid ( Sheppard, R.C. et al., J.Chem. Soc. Chem. Comm., pp. 165-166 (1985)), using an Advanced Chemtech mode! 200 available from Advanced Chemtech., Louisville, Ky., using a Millipore 9050+ available from Millipore, Bedford Mass, or other available instrumentation.
T20 may be produced by incorporating cDNA coding compounds of the invention into functional viral or circular plasmid DNA vectors. The vectors or plasmids can be used to transfect or transform selected microorganisms. The transformed or transected microorganisms can be cultured under conditions that are conducive to express vector-borne DNA sequences and isolation of the desired peptides from the growth medium can be achieved. (See, for example United States Patent No. 5,955,422, the entirety of which is incorporated herein by reference as if recited in full.)
T20 may also be prepared by standard recombinant DNA technology using techniques that are well known in the art. For example, the procedures outlined in Sambrook et al. Molecular Cloning: A Laboratory Manual, 2nd edition, (Cold SpR1ng Harbor Press, Cold SpR1ng Harbor, N.Y.) or Ausubel et a!., Current Protocols in Molecular Biology . John Wiley and Sons, New York (1995), both of which are herein incorporated by reference.
A particular method for making T20 is descR1bed in U.S. Patent, 6,015,881 which is hereby incorporated.

After cleavage and deportation, T20 may be puR1fied by any suitable means. For example, ion exchange, gel filtration chromatography and/or a reverse-phase column/HPLC system can be used to puR1fy full length T20 from fragments thereof. In the case when a T20 precursor is first prepared, with a blocking/protecting group attached to the N-terminus (e.g., acyl group) and/or the C-terminus {e.g., amino group), one or both of those groups may be removed using known techniques.
The amino acid sequence of T20 may be confirmed and identified using standard amino acid analysis as well as manual and automated Edman degradation and determination of each amino acid. HPLC analysis and mass spectrometry may also be used to veR1fy the production of T20.
Polyethylene glycol aldehyde compounds which may be reacted with T20 nnay also be made in any desired manner. It is preferred, however, that the polyethylene glycol be made in accordance with the methods descR1bed in concurrently filed U.S. Patent Application SeR1al Number 60/398,196 filed July 24, 2O02 entitled "Polyethylene Glycol Aldehydes," the entirety of which is hereby incorporated by reference.
Generally, a polyethylene glycol aldehyde of the formula:

wherein R1, m, n, and p are defined as above is used to pegylate the T20. The polyethylene glycol aldehyde used to pegylated the T20 may be prepared by any suitable means. One preferred polyethylene glycol aldehyde is prepared as follows:

Polyethylene glycol aldehydes of varying size {e.g., varying n values) may be prepared by following the general reaction scheme above.

The pegylated T20 compounds of the present invention may be prepared by any suitable means. Further provided by the invention, however, is a method for pegylating a T20 polypeptide compR1sing reacting a T20 polypeptide, NHT20, with a polyethylene glycol aldehyde of formula:

wherein R1, m, n, n, and p are defined as above; to produce a compound of formula:

wherein the polyethylene glycol aldehyde molecule is bonded to the N-terminal amino group of the T20 polypeptide.
The pegylated T20 is prepared by adding T20 and the PEG reagent in a molar ratio range of 1:1 to 1:100. The T20 has a free a-amino group (any acyl group is removed) and either a free carboxy group or an amino-protected carboxy group, as discussed above. The reaction mixture is placed in a borate, phosphate, or tR1 buffer at room temperature or 4 degrees Celsius for about .5 to 24 hours at a pH range of 5.5 to 7.4. The molar ratio of PEG reagent to peptide/proteins is between 1:1 to 100:1. The concentration of peptide/proteins is between 1 to 10 mg/ml. The concentration of buffer is usually 10 to 500 mM.
The pegylated T20 is puR1fied by taking the reaction mixture of pegylated T20 " and diluting it with an equilibration buffer (20mM TR1s, pH 7.5). The resulting mixture is then applied on a Q-Sepharose column. After the mixture is applied on

the QA column, it is washed with the equilibration buffer eluted with 75 M NaCl; eluted with 200 mM NaCI; eluted with 1M NaCl; and regenerated with 1M HOAC + 1M NaCl and 0.5 NaOH.
By using reverse phase HPLC, a it is possible to readily separate and isolate the N-terminal, monopegylated product from other byproducts in the mixture.
In preferred embodiments of the pegylated T20 polypeptides of the present invention, p is 3, R1 is methyl, m is 1, and n is from 100 to 750; or p is 2, R1 is methoxy, m is 1, and n is from 100 to 750; or p is 1, R1 is methoxy, m is 1, and n is from 100 to 750.
The present invention also provides a pegylated T20 polypeptide of the following formula:

wherein n is from 10 to 1,000 and NHT20 is a T20 polypeptide covalently bonded through its terminal a-amino group. In one embodiment n is approximately 225, for example 227.
This pegylated T20 polypeptide may be made in any desired manner, preferably, it is made by the method descR1bed in Example 3.

The pharmaceutical compositions of the invention comprise, in admixture with a pharmaceutically acceptable excipient, a compound of formula (1),
wherein R1, m, n, p, and NHT20 are defined as above.
The pharmaceutical compositions of the present invention comprising pegylated T20 polypeptides or the salts thereof, may be manufactured in any desired manner, e.g., by means of conventional mixing, encapsulating, dissolving, granulating, emulsifying, entrapping, or lyophilizing processes. These pharmaceutical preparations may be formulated with therapeutically inert, inorganic or organic excipients and carriers. Suitable excipients for injection include water, alcohols, polyols, glycerine, vegetable oils, phospholipids and surfactants.
The pharmaceutical preparations may also contain preserving agents, solubilizing agents, stabilizing agents, wetting agents, emulsifying agents, sweetening agents, coloring agents, flavoring agents, salts for varying the osmotic pressure, buffers, coating agents, or antioxidants. They may also contain other therapeutically valuable substances, including additional active ingredients.
The formulations suitable for parenteral (including subcutaneous, intramuscular, intravenous, intradermal, intratracheal, and epidural) administration may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of bringing into association the pegylated T20 polypeptides and the pharmaceutical carrier(s) or excipient(s). In general, the formulations are prepared by uniformly and intimately bringing into association the pegylated T20 polypeptides with liquid

carriers. Formulations suitable for parenteral administration include: aqueous and non-aqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents. The formulations may be presented in unit-dose or multi-dose containers, for example, sealed ampoules and vials, and may be stored in a freeze-dried (lyophilized) conditions requiring only the addition of the sterile liquid carrier, for example, water for injections, immediately prior to use.
Preferred unit dosage formulations are those containing a daily dose or unit, daily sub-dose, as herein above recited, or an appropriate fraction thereof, of the administered ingredient.
Preferably, the pegylated T20 polypeptide is in unit dosage form. As used herein, "unit dosage form," means that an amount appropriate for a single dose the pegylated T20 polypeptide is in a premature and/or prepackaged form. This allows for convenient preparation of the pegylated T20 polypeptide for administration, and may even allow for self-administration by the patient. The unit dosage amount will obviously depend on the amount of pegylated T20 polypeptide to be delivered, and the frequency of dosing.
The pegylated T20 polypeptide may also be provided in a lyophilized powder form in a unit dosage amount, suitable for reconstitution with a pharmaceutically acceptable excipient just prior to the time of administration.

A particular pharmaceutical composition of the invention comprises, in admixture with a pharmaceutically acceptable excipient, a compound of formula (I),
wherein R1 is methoxy, m is 1, n is from 100 to 750, and p is 3.
Another pharmaceutical composition of the invention is a pharmaceutical composition comprising, in admixture with a pharmaceutically acceptable excipient, a compound of formula (III),
wherein n is from 10 to 1,000 and NHT20 is a T20 polypeptide covalently bonded through its terminal a-amino group. In one embodiment n is approximately 225, for example 227.
The present invention further provides methods of inhibiting HIV infection comprising administering to a patient a pharmaceutical composition comprising, in admixture with a pharmaceutically acceptable excipient, a compound of formula (I),
wherein Ri, m, n, p, and NHT20 are defined as above.
The pegylated T20 polypeptides are generally administered in the manner (unlegislated) T20 polypeptides are presently administered. Modifications may be made, however, to take advantage of the improved pharmacokinetic properties of the pegylated T20 polypeptides.
In the method of inhibiting HIV of the invention, the pharmaceutical composition may be administered in any suitable manner and route. In a preferred

method the pegylated T20 polypeptide is administered in the form of an injectable solution or suspension. Preferably, the injectable solution or suspension is administered by subcutaneous injection or intravenously.
In another preferred method, the pegylated T20 polypeptide is administered though a transdermal delivery device, e.g., a transdermal patch.
In the method of inhibiting HIV of the invention, the pharmaceutical composition may be administered in any suitable dosage and schedule. The pharmaceutical compositions of the invention can be administered in any form, and via any route, desired. Generally, however, the pegylated T20 polypeptides of the present invention are administered parent rally, for example, in the form of injection solutions.
Determination of a therapeutically effective amount is within the skill in the art, and the therapeutically effective amount or dosage of a pegylated T20 polypeptide according to this invention may vary and will be adjusted to the individual requirements in each particular case. In general, in the case of parenteral administration to adult humans weighing approximately 70 Kg, a daily dosage of about 5 mg to about 300 mg, preferably from about 50 mg to about 200 mg, should be appropriate, although the upper limit may be exceeded when indicated. The dosage may be administered as a single dose, in divided doses, or as continuous infusion. Daily and, more preferably, weekly administrations may be employed.

The present invention also provides a method of inhibiting HIV infection comprising administering a pharmaceutical composition comprising, in admixture with a pharmaceutically acceptable excipient, a compound of formula (I),
wherein R1 is methoxy, m is 1, n is from 100 to 750, and p is 3.
Also contemplated within the scope of the invention is a method of inhibiting HIV infection comprising administering a pharmaceutical composition comprising, in admixture with a pharmaceutically acceptable excipient, a compound of formula (111),
wherein n is from 10 to 1,000 and NHT20 is a T20 polypeptide covalently bonded through its terminal a-amino group. In one embodiment n is approximately 225, for example 227.
The following examples are provided to further illustrate the compounds, compositions, and methods of the present invention. These examples are illustrative only and are not intended to limit the scope of the invention in any way.
Example 1
Preparation of PEG10K-butanoaldehyde
mPEG of molecular weight 10,000 (30.0 g, 3 mmol) in 240 mL of toluene was azeotropically dried by refluxing for 2 hours, followed by the removal of 120 mL of toluene. The resulting solution was cooled to room temperature then potassium

tert-butoxide (0.68 g, 6 mmol) in 20 ml of absolute tert-butanol and 20 ml of toluene was added to the PEG solution. The resulting mixture was stirred for two hours at room temperature under argon. Tert-butyl bromoacetate (1.00 mL, 6.75 mmol) was added to the reaction via syringe and the reaction was stirred overnight at room temperature under argon. The reaction solution was then condensed by rotary evaporation. The residue was precipitated by addition to diethyl ether. The precipitated mPEGiok t-butyl carboxymethyl ester product was filtered off and dried in vacuo. Yield: 28 g. NMR (de-DMSO): 1.40 ppm (t, 9H, -CH3); 3.21 ppm (s, -

mPEG10k t-butyl carboxymethyl ester (26.5 g) was then dissolved in 350 mL of 1N sodium hydroxide and the solution was stirred at room temperature overnight. The pH of the mixture was adjusted to 2.5 by addition of 6 N hydrochloric acid, and the mixture was extracted with dichloromethane. The organic layer was dried over sodium sulfate, filtered, concentrated, and precipitated into diethyl ether. The product m-PEG10k-carboxymethyl acid was collected by filtration and dried under vacuum. Yield: 24 g. NMR (de-DMSO): 3.21 ppm (s, -OGH3); 3.5 ppm (s, -O-CH2CH2-O-); 3.99 ppm (s, 2H, -O-CH2-COOH).
mPEGiok-carboxymethyl acid (6 g, 0.6 mmol) was then dissolved in anhydrous dichloromethane (30 mL) followed by the addition of 4-aminobutylraldehyde diethylacetal (140 ml, 0.9 mmol), 1-hydroxybenzotriazole (80 mg, 0.6 mmol), and dicyclohexylcarbodiimide (160 mg, 0.78 mmol). The mixture was stirred overnight at room temperature under argon. The reaction mixture was filtered, concentrated, and precipitated with mixture of 2-propanol and diethyl ether

(1:1). The mPEGiok-butanoacetal product was dried in vacuo overnight. Yield: 5.4 g. NMR (de-DMSO): 1.07-1.12 ppm (t, 6H, (-0-CH2-CH3)2); 1-46 ppm (m, 4H, -NHCH2CH2CH2-CH-); 3.08-3.11 ppm (q, 2H, -NHCH2CH2CH2-CH-); 3.21 ppm (s, -OCH3); 3.5 ppm (s, -O-CH2CH2-O-); 3.85 ppm (s, 2H, -O-CH2-CO-NH-); 4.44 ppm (t, 1H, -NHCH2CH2CH2-CH-); 7.67 ppm (-NH-).
mPEG10k-butanoacetal (2 g, 0.2 mmo!) was then dissolved in 20 mL of 80 % CF3COOH and the solution was stirred at room temperature overnight. The pH of the mixture was adjusted to 6.0 by addition of 1 N NaOH solution, and sodium chloride (10 wt %) was added and then the pH of the solution was adjusted to 7.0 by addition of 1 N NaOH. The mixture was extracted with dichloromethane. The organic layer was dried over sodium sulfate, filtered, concentrated, and precipitated into diethyl ether. The product mPEGiok-butanoaldehyde was collected by filtration and dried under vacuum. Yield: 1.7 g. NMR (ds-DMSO): 3.21 ppm (s, -OCH3); 3.5 ppm (s, -O-CH2CH2-O-); 3.85 ppm (s, 2H, -O-CH2-CO-NH-); 7.67 ppm (-NH-); 9.66 ppm (-CHO-).
Example 2
Peqvlation of T20 with PEGioK-butanoaldehyde
Butanoaldehyde of PEG lOkDa (from Example 1) was added to 15 mg of T20 (purity 93.7 %) in 3.0 ml of buffer (50 mM potassium phosphate pH 6.5) in a molar ratio of 5 moles of reagent per one mole of T20. The T20 polypeptide was deacylated at the a-amino terminus, but protected at the carboxyl terminus by -NH2. To the reaction mixture 10% (V/V) of 0.5 M sodium cyanoborohydride solution in

water was added and stirred for 4 hours at room temperature. Pegylated T20 was purified from the reaction mixture using ion exchange chromatography (QA). A linear gradient with increasing salt concentrations from 150 mM to 1 M NaC! in 20 mM Tris, pH 7.5 was used to separate pegylated T20 and unmodified T20.
Example 3 Peqylation of T20 with mPEG10k-propionaldehyde
; A propionaldehyde of PEG lOkDa, having the following structure is used.

150 mg of mPEGIOk-propionaldehyde was added to 15 mg of T20 (purity 93.7 %) in 3.0 ml of buffer (50 mM potassium phosphate pH 6.5) in a molar ratio of 5 moles of reagent per one mole of T20. The T1249 polypeptide was deacylated at the a-amino terminus, but protected at the carboxyl terminus by -NH2.
To the reaction mixture 10% (V/V) of 0.5 M sodium cyanoborohydride solution in water was added and stirred for 4 hours at room temperature. Pegylated T20 was purified from the reaction mixture using ion exchange chromatography (QA). The structure of the pegylated T20 polypeptide follows;

A linear gradient with increasing salt concentrations from 150 mM to 1 M NaCl in 20 mM Tris, pH 7.5 was used to separate pegylated T20 and unmodified T20.

Example 4 Inhibitor Concentration for Pegylated T20
Phenotypic susceptibility is usually quantified in terms of IC50 or IC90 a measure of the concentration of drug needed to inhibit 50% or 90%, respectively, of viral growth.
Inhibitor concentration 50 and inhibitor concentration 90 results for the pegylated T20 with PEG10K-propionaldehyde (Example 3) and pegylated T20 with mPEGIOk-butanoaldehyde (Example 2) has been reproduced in Table 1 below:

Example 5 MAGI/MAG Antiviral Assays
These assays score for reduction of infectious virus titer employing tine indicator cell lines MAG! (Multinuclear Activation of a Galactosidase lndicator)or the CCRS-expressing derivative cMAGI. The MAGI cell line was derived from parental HeLa cells by introducing genes for CD4 and an HlV-1 LTR-driven b-gal reporter with an amphotropic retrovirus vector (Kimpton J, Emerman M, J Virol 66:2232-9, 1992). The cMAGI cell line was derived from the MAGI cell line by introduction of the OCRS gene using the amphotropic retroviral vector, PA317 (Chackerian B, Long EM, Luciw PA, Overbaugh J, J Virol 71:3932-9, 1997.). The cMAGI cells support replication of primary NSI (R5) isolates and laboratory adapted X4 viruses, while the MAGI cells support replication of only X4 viruses. Both cell lines exploit the ability of HIV-1 tat to transactivate the expression of a b-galactosidase reporter gene driven by the HIV-LTR. The b-gal reporter has been modified to localize in the nucleus and can be detected with the X-gal substrate as intense nuclear staining within a few days of infection. The number of stained nuclei can thus be interpreted as equal to the number of infectious virions in the challenge inoculum if there is only one round of infection prior to staining.
An inhibitor of infection and cell-cell fusion,e.g., T20 or T1249 (Wild C, Greenwell T, Matthews T, AIDS Res Hum Retroviruses 9:1051-3, 1993), was added 24 hrs post-infection in order to permit a readout that confidently represents

a single round of infection. Infected cells were enumerated using a CCD-imager and both primary and laboratory adapted isolates showed a linear relationship between virus input and the number of infected cells visualized by the imager. In the MAGI and cMAGl assays a 50% reduction in infectious titer (VnA/o = 0.5) is significant and provides the primary cutoff value for assessing antiviral activity. A 90% reduction in infectious titer (VNNo) is used as an additional cutoff value on assessing antiviral activity.
Each test compound dilution was tested in duplicate against a virus inoculum adjusted to yield approximately 1500-2000 infected cells/well of a 48 well micro titer plate. The test compound was added to the cMAGI or MAGI cells, followed by the virus inocula, and 24 hrs later, an inhibitor of infection and cell-cell fusion (Wild C, Greenwell T, Matthews T, AIDS Res Hum Retroviruses 9:1051-3, 1993) was added to prevent secondary rounds of infection and cell-cell virus spread, The cells were cultured for 2 more days, fixed and stained with the X-gal substrate to detect infected cells. The number of infected cells for each control and test compound dilution were determined with the CCD-imager. IC50 is defined as the dilution of a text compound resulting in a 50% reduction in infectious virus titer. IC90 is defined as the dilution resulting in a 90% reduction in infectious titer.
The invention being thus described, it will be obvious that the same may be
r
varied in many ways. Such variations are not to be regarded as a departure from the spirit and scope of the invention and all such modifications are intended to be included within the scope of the following claims.

WE CLAIM :
A compound of formula (I):
wherein
Ri is a capping group,
mis from 1 to 17,
n is from 10 to 1,000,
pis from 1 to 3, and
NHT20 is a T20 polypeptide covalently bonded through its terminal a-amino group.
2. A compound according to claim 1, wherein Ri is selected from the
group consisting of halogen, epoxide, maleimide, orthopyridyl disulfide, tosylate,
isocyanate, hydrazine hydrate, cyanuric halide, N-succinimidy!oxy, sulfo-N-
succinimidyloxy, 1-benzotriazolyloxy, 1-(midazolyloxy, p-nitrophenyloxy, and

3, A compound according to claim 1, wherein p is 3.

4. A compound according to claim 1, wiierein p is 3, R1 is methoxy, m is
1, and n is from 100 to 750.
5. A compound of formula:
wherein n is from 10 to 1,000 and NHT20 is a T20 polypeptide covalently bonded tlirough its terminal a-amino group.
6. A pharmaceutical composition comprising, in admixture with a
pharmaceutically acceptable excipient, a compound of formula:

wherein
R1 is a capping group,
m is from 1 to 17,
n is from 10 to 1,000,
p is from 1 to 3, and
NHT20 is a T20 polypeptide covalently bonded through its terminal a-amino group.
7. A pharmaceutical composition according to claim 6, wherein p is 3, R1
is methoxy, m is 1, and n is from 100 to 750.

8. A pharmaceutical composition according to claim 6 in the form of a
lypholized powder.
9. A pharmaceutical composition according to claim 6 in the form of an
injectable solution or suspension,
10. A process for attaching a polyethylene glycol molecule to a T20
polypeptide comprising reacting a T20 polypeptide with a poiyethylene giyco!
aldehyde of formula:
wherein
R is a capping group,
m is from 1 to 17,
n is from 10 to 1,000, and
p is from 1 to 3; to produce a compound of formula:
R1(CH2CH2O)n-CH2CH2-O-(CH2)m-CO-NH-(CH2)p-CH2-NHT20 (1) wherein the polyethylene glycol aldehyde molecule is bonded to the N-terminai amino group of the T20 polypeptide.

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Patent Number

214374

Indian Patent Application Number

3122/CHENP/2004

PG Journal Number

13/2008

Publication Date

31-Mar-2008

Grant Date

11-Feb-2008

Date of Filing

31-Dec-2004

Name of Patentee

F. HOFFMANN-LA ROCHE AG

Applicant Address

124 Grenzacherstrasse, CH-4070 Basel,

Inventors:

#	Inventor's Name	Inventor's Address
1	BAILON, Pascal, Sebastian	21 Woodbine Road, Florham Park, NJ 07932,
2	WON, Chee-Youb	91 Belmont Drive, Livingston, NJ 07039,

PCT International Classification Number

C07K 14/16

PCT International Application Number

PCT/EP2003/007710

PCT International Filing date

2003-07-16

PCT Conventions:

#	PCT Application Number	Date of Convention	Priority Country
1	60/398,195	2002-07-24	U.S.A.