HK1082953A - Pegylated t20 polypeptide - Google Patents

Description

Pegylated T20 polypeptide

no marking

The present invention relates to pegylated T20 polypeptide compounds, and to methods of using and making such compounds, e.g., in pharmaceutical compositions, and to therapeutic methods of performing therapy.

Some viruses, particularly HIV, must undergo a complex process called fusion to enter host cells and propagate. During the fusion process, the outer membrane of the virus fuses with the cell membrane of the host cell. In the case of HIV, the HIV virus envelope and CD during reproduction₄ ⁺Cell membrane fusion of T cells.

T20 is a member of a new class of antiviral agents that inhibit viral/cell membrane fusion. In thatIn the case of HIV, it provides two beneficial effects: blocking reproduction of HIV and no CD induced thereby₄ ⁺The T cells die.

Data from two large international phase III clinical trials indicate that the combination of T-20 in therapy reduces HIV in the blood of at least two-fold percentage of patients to undetectable levels and provides an enhanced immune response at 24 weeks compared to combination therapy not receiving T-20. In addition, those receiving T-20 are less likely to experience virological failure or relapse during the 24 week period.

In the first phase III clinical trial in north america and brazil, 37% of patients presented undetectable levels of HIV in the blood at 24 weeks (less than 400 copies/ml) when treated with T-20 in combination with the optimized basal regimen, as opposed to receiving only 16% of the basal regimen alone (p < 0.0001). Combination therapy with T-20 further reduced the HIV viral load to less than 50 copies/ml in 20% of patients, while only 7% of patients treated with combination therapy alone (p ═ 0.0002).

The initial efficacy endpoint of this study, in which the mean difference in the magnitude of reduction of HIV between the two groups was 0.934log10 copies/ml (p < 0.0001). The HIV level in patients treated with T20 as part of the combination regimen was reduced by 1.697log10 copies/ml compared to 0.763log10 copies/ml in the control group. Furthermore, HIV levels were reduced by 1.0log10 or more in 52% of patients receiving T-20, compared to 29% in patients not receiving T-20 (p < 0.0001). Patient CD in T20 group₄ ⁺The average cell rise was 76 cells/mm compared to 32 cells/mm in the control (p < 0.0001).

The results of the second phase three clinical trial conducted in europe and australia were consistent with the findings of the initial study. Of the patients treated with the T-20 combination optimized basal regimen, 28% of the patients had undetectable levels of HIV in the blood at 24 weeks (less than 400 copies/ml), while 14% of the patients treated with the optimized basal regimen alone (p < 0.0001). Combination therapy with T-20 further reduced the HIV viral load to less than 50 copies/ml in 12% of patients, while 5% of patients receiving combination therapy alone (p-0.0099).

The mean difference in the magnitude of HIV reduction between the two groups at 24 weeks was 0.78log10 copies/ml (p < 0.0001). HIV levels in patients receiving T-20 as part of the combination regimen were reduced by an average of 1.43log10 copies/ml, as compared to an average of 0.65log10 copies/ml in the control group. Furthermore, HIV levels were reduced by 1.0log10 or more in 43% of patients receiving T-20, compared to 21% in patients not receiving T-20 (p < 0.0001). Patient CD in T-20 group₄ ⁺The cells increased on average 65 cells/mm, whereas the control group had 38 cells/mm (p ═ 0.023).

Initially, based on the treatment history and the tests for retroviral resistance, an optimized basic regimen (consisting of 3 to 5 drugs, including up to two newly approved or newly studied drugs if appropriate) was selected for each patient. After the regimen was selected, patients were randomized into 2: 1 groups and received either a combination treatment regimen with T-20 or a basal regimen alone. Patients randomized to the T-20 group injected subcutaneously twice daily with 90mg of T-20.

In current clinical treatment of HIV, the development of viral resistance to newly approved anti-HIV drugs is a significant problem. Over time, many patients who begin to be treated with a combination of newly approved antiretroviral drugs develop resistance to one or more of these drugs. However, studies have shown that T-20 is not affected by resistance to any of the currently approved antiretroviral drugs. (the data is presented at 6 months 4-8 days 2001 at the 5 th international conference on drug resistance and treatment strategies held by Stokes, Arizona). Other experiments showed that the in vitro activity of T20 was not affected by mutations associated with reverse transcriptase inhibitor and protease inhibitor resistance.

Like many polypeptide therapeutics, T20 is typically administered by injection. Current treatment methods typically involve injections more than once a day.

Therefore, it would be advantageous to provide T20 polypeptides and pharmaceutical compositions having superior performance and pharmacokinetic properties. T20, which provides lower T20 therapeutic doses, lower frequency of administration, and/or has an extended duration of action, is particularly advantageous.

These and other objects of the present invention are described in more detail below.

The present invention provides compounds of the formula:

wherein

R₁Is a terminal group, and is a terminal group,

m is the number of 1 to 17,

n is 10 to 1,000,

p is from 1 to 3, and

NHT20 is a T20 polypeptide covalently linked through its terminal alpha-amino group.

In one embodiment of the compounds of the invention, R₁Is methoxy, m is 1, n is 100-750, and p is 3.

Also provided is a pharmaceutical composition comprising a compound of formula (I) wherein R is in admixture with a pharmaceutically acceptable excipient₁M, n, p and NHT20 are as described above.

In one embodiment of the pharmaceutical composition of the invention, R₁Is methoxy, m is 1, n is 100-750, and p is 3.

The present invention also provides a method of inhibiting HIV infection comprising administering a pharmaceutical composition comprising a compound of formula (I) wherein R is in a compound of formula (I)₁M, n, p and NHT20 are as described above.

In one embodiment of the method of inhibiting HIV infection, R₁Is methoxy, m is 1, n is100, 750, and p is 3.

If the present invention relates to the "method for preparing …", this is referred to as "procedure for preparing …".

Also provided are methods of making a pegylated T20 polypeptide, comprising reacting a T20 polypeptide with a polyethylene glycol aldehyde of the formula:

wherein R is₁M, N and p are as described above to produce a compound of formula (I) wherein a polyethylene glycol aldehyde molecule is attached to the N-terminal amino group of a T20 polypeptide.

If the present invention relates to a "method for inhibiting HIV infection comprising a compound," it means "using the compound for the manufacture of a medicament for inhibiting HIV".

The invention also provides compounds of the formula

CH₃-O-(CH₂-CH₂-O)_n-CH₂-CH₂-O-CH₂-CH₂-CH₂-NHT20(III)

Where n is 10-1,000, NHT20 is a T20 polypeptide covalently linked through its terminal alpha-amino group.

The invention also provides a pharmaceutical composition comprising a compound of formula (III) wherein n is 10-1000 and NHT20 is a T20 polypeptide covalently linked through its terminal alpha-amino group, in admixture with a pharmaceutically acceptable excipient.

Also provided are methods of inhibiting HIV infection comprising administering a pharmaceutical composition comprising a compound of formula (III) wherein n is 10-1000 and NHT20 is a T20 polypeptide covalently linked through its terminal alpha-amino group, in admixture with a pharmaceutically acceptable excipient.

As mentioned above, T20 is a "fusion inhibitor" polypeptide. T20 consists of 36 amino acids. The polypeptide sequence of T20 is:

YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF[SEQ.ID.NO：1]

the N-terminal (or amino-terminal) amino acid is tyrosine (Y) and the C-terminal (or carboxy-terminal) amino acid is phenylalanine (F).

As shown in FIG. 1 of U.S. Pat. No. 5,464,933(SEQ ID: 1), which is incorporated herein by reference in its entirety, the T20 polypeptide sequence may be blocked/derivatized at one or both of its amino-and carboxy-termini. As described in U.S. Pat. No. 5,464,933, the amino terminus of tyrosine can be blocked/derivatized with an acyl group and the carboxy terminus of phenylalanine can be blocked/derivatized with an amino group (the latter resulting in-COOH to-CONH)₂Transformation of (d).

As used herein, "T20" is to be understood as [ seq.id no: 1]Optionally blocked with an amino group at the C-terminus of phenylalanine. That is, when referring to "T20", the phenylalanine C-terminus is either-COOH or CONH₂。

The present invention provides pegylated T20 compounds of the formula:

wherein

R1 is a terminal group,

m is the number of 1 to 17,

n is 10 to 1,000,

p is 1-3, and

NHT20 is a T20 polypeptide covalently linked through its terminal alpha-amino group.

As used herein, R₁The "end capping group" may be any suitable chemical group, which is generally reactive or non-reactive with other chemical components, depending on preference. In the above compounds, polyethylene glycol was covalently linked to the α -amino group of T20. Selection of R₁The end capping group is intended to permit or prevent bifunctional, e.g., covalent attachment to a second chemical moiety of interest.

In the case where the end capping group is not generally reactive with other chemical moieties, R₁Is relatively inert and therefore does not covalently link to another chemical component. Generally suitable unreactive R₁The end capping groups include: hydrogen, hydroxy, lower alkyl, lower alkoxy, lower cycloalkyl, lower alkenyl, lower cycloalkenyl, aryl and heteroaryl.

As used herein, the term "lower alkyl" refers to a substituted or unsubstituted, straight or branched chain alkyl group containing 1 to 7 carbon atoms, preferably 1 to 4 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, n-pentyl, n-hexyl, n-heptyl, and the like.

The term "lower alkoxy" means a lower alkyl group as defined above attached through an oxygen atom, and examples of lower alkoxy are methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, tert-butoxy, n-pentoxy and the like.

The term "lower cycloalkyl" refers to a substituted or unsubstituted cycloalkyl group containing 3 to 7, preferably 4 to 6, carbon atoms, i.e. cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl or cycloheptyl.

As used herein, the term "lower alkenyl" refers to substituted or unsubstituted straight or branched chain alkenyl groups containing from 2 to 7, preferably from 2 to 5, carbon atoms, such as vinyl, butenyl, pentenyl, hexenyl and the like.

The term "lower cycloalkenyl" refers to substituted or unsubstituted cycloalkenyl groups containing 4-7 carbon atoms, e.g., cyclobutenyl, cyclopentenyl, cyclohexenyl, and the like.

The term "aryl" refers to phenyl or naphthyl which is unsubstituted or optionally mono-or polysubstituted with halogen, lower alkyl, lower alkoxy, trifluoromethyl, hydroxy, carboxylic acid, carboxylic ester, nitro, amino or phenyl, especially with halogen, lower alkyl, lower alkoxy, trifluoromethyl, hydroxy, nitro, amino and phenyl.

The term "heteroaryl" refers to a 5 or 6 membered heteroaryl group containing one or more heteroatoms selected from N, S and O, which may also be benzo and/or substituted in the same manner as the "aryl" group described previously.

Generally preferred non-reactive R₁The end capping group comprises methoxy, hydroxy or benzyloxy. Particularly preferred R₁The end capping group is methoxy. When R is₁In the case of methoxy, the pegylated polypeptide compound is sometimes referred to herein as a "mPEG" compound, wherein m represents methoxy.

If R is₁The end capping group is typically reactive with other chemical moieties, then R₁I.e. functional groups that are reactive with functional groups such as amines and/or thiols in peptides and/or proteins. In this case, R₁May be a functional group that readily reacts with electrophilic or nucleophilic groups on other molecules, as opposed to those groups that require strong catalysts or harsh reaction conditions to react. If R is₁Is relatively active, the polyethylene glycol aldehyde may be covalently linked to another chemical component.

Generally appropriate reactivity R₁Examples of end capping groups include: halogen, epoxide, maleimide, ortho-pyridine disulfide (ortho-pyridine disulfide), tosylate, isocyanate, hydrazine hydrate, cyanuric halide, N-succinimidyloxy, thio-N-succinimidyloxy, 1-benzotriazolyloxy, 1-imidazolyloxy, p-nitrophenyloxy and

the term halogen means fluorine, chlorine, bromine or iodine. Reactive R is generally preferred₁The end capping group is.

When R is present₁When a blocking group is present, it is understood that in the compounds of the invention, the first m, n and/or p in the formula may be the same or different from the second m, n and/or p. However, it is preferred that both m have the same value, both n have the same value and both p have the same value.

In the present invention, m is 1 to 17. In a preferred embodiment, m is from 1 to 14. More preferably m is from 1 to 7, even more preferably m is from 1 to 4, and most preferably m is 1.

In the present invention, n is 10 to 1,000. In a preferred embodiment of the 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, and most preferably n is 100-1,000.

In the present invention, p is 1 to 3. Preferably p is 3.

In a preferred embodiment p is 3, R₁Is methoxy, m is 1, n is 100-750; or p is 2, R₁Is methoxy, m is 1, n is 100-750; or p is 1, R₁Is methoxy, m is 1, n is 100-750.

The invention provides embodiments of formula (I) wherein R₁Is methoxy, m is 1, n is 100-750, p is 3, NHT20 is a T20 polypeptide covalently linked through its terminal alpha-amino group.

As mentioned above, the PEGylated T20 compounds of the present invention covalently attach the alpha-amino group of T20 to a polyethylene glycol derivative of specific structure. Pegylated compounds can be produced by any desired means, but are generally prepared by reacting T20 with a separately prepared polyethylene glycol derivative.

The T20 polypeptide may be prepared in any suitable manner. For example, the compound can be synthesized by conventional Merrifield solid phase synthesis techniques involving the use of Boc-amino acids in the solid phase (chem.Soc., 85, 2149, 1963) by manual or automated procedures, and Fmoc-amino acids in the solid phase (Sheppard, R.C., et al, J.chem.Soc.chem.Comm., 165-Page 166 (1985)), using Advanced Chemtech model 200, purchased from Advanced Chemtech, Louisville, Ky., and Millipore9050, purchased from Millipore, Bedford masses⁺Or other useful instrumental synthesis.

T20 can be produced by introducing a cDNA encoding a compound of the invention into a functional viral or circular plasmid DNA vector. The vector or plasmid may be used to transfect or transform a selected microorganism. The transformed or transfected microorganism is cultured under conditions conducive to the DNA carried by the expression vector, and the desired polypeptide can then be isolated from the growth medium (see, e.g., U.S. Pat. No. 5,955,422, incorporated herein by reference in its entirety).

T20 can also be prepared by standard recombinant DNA techniques using techniques well known in the art. For example, the method described by Sambrook et al, Molecular Cloning: a laboratory Manual, second edition, (Cold Spring Harbor Press, Cold Spring Harbor, N.Y.) or Ausubel et al, Current Protocols in Molecular Biology, John Wiley and Sons, New York (1995), which are incorporated herein by reference.

The specific method of producing T20 is set forth in U.S. patent No. 6,015,881, incorporated herein by reference.

T20, after dissociation and deprotection, may be purified by any suitable means. For example, ion exchange, gel filtration chromatography and/or reverse phase column/HPLC systems may be used to purify full length T20 from its fragments. In the case where the T20 precursor was originally prepared with a blocking or protecting group attached to the N-terminus (e.g., acyl) and/or a blocking or protecting group attached to the C-terminus (e.g., amino), one or both of these groups can be removed using known techniques.

The amino acid sequence of T20 can be confirmed and identified for each amino acid using standard amino acid analysis and manual and automated Edman degradation. HPLC analysis and mass spectrometry can also be used to confirm the production of T20.

The polyethylene glycol aldehyde compound capable of reacting with T20 can also be produced in any desired manner. However, it is preferred that the polyethylene glycol be produced according to the method described in U.S. patent application Ser. No. 60/398,196 filed concurrently at 24.7.2002, entitled "polyethylene glycol aldehyde", which is hereby incorporated by reference in its entirety.

Typically, a polyglycol aldehyde of the formula is used to PEGylate T20

Wherein R is₁M, n and p are as described above. The polyethylene glycol aldehyde used to pegylate T20 may be prepared by any suitable means. One preferred polyethylene glycol aldehyde is prepared as follows:

mPEG_10kbutyraldehyde reaction scheme

Polyethylene glycol aldehydes of different sizes (e.g., different values of n) can be prepared according to the reaction scheme above.

The pegylated T20 compounds of the invention can be prepared by any suitable means. The invention also provides a method of pegylating a T20 polypeptide comprising reacting a T20 polypeptide NHT20 with a polyethylene glycol aldehyde of the formula:

wherein R is₁M, n, p are as described above to produce a compound of the formula:

R₁-(CH₂CH₂O)_n-CH₂CH₂-O-(CH₂)_m-CO-NH-(CH₂)_p-CH₂-NHT20 (I)

wherein the polyethylene glycol aldehyde molecule is connected with the N-terminal amino group of the T20 polypeptide.

Pegylated T20 was prepared by adding T20 and polyethylene glycol reagents in a molar ratio of from 1: 1 to 1: 100. As discussed above, T20 has a free α -amino group (minus all acyl groups) and a free carboxyl group or an amino protected carboxyl group. The reaction mixture is placed in borate, phosphate or tri buffer at a pH in the range of 5.5-7.4 for about 0.5 to 24 hours at room temperature or 4 ℃. The molar ratio of polyethylene glycol reagent to peptide/protein is between 1: 1 and 100: 1. The peptide/protein concentration is 1-10 mg/ml. The concentration of the buffer is usually 10 to 500 mM.

Pegylated T20 was purified by removing the pegylated T20 reaction mixture and then diluting with equilibration buffer (20mm tris, pH 7.5). The resulting mixture was passed through a Q-Sepharose column. After the resulting mixture was added to the QA column, it was washed with an equilibration buffer; eluting with 75M NaCl; 200mM NaCl elution; eluting with 1M NaCl; and regenerated with 1M HOAC +1M NaCl and 0.5 NaOH.

The N-terminally monopegylated product can be easily separated and isolated from the other by-products in the mixture by using reverse phase HPLC.

In a preferred embodiment of the pegylated T20 polypeptides of the invention, p is 3 and R₁Is methyl, m is 1, n is 100-750; or p is 2, R₁Is methoxy, m is 1, n is 100-750; or p is 1 or a group of compounds,R₁is methoxy, m is 1, n is 100-750.

The invention also provides pegylated T20 polypeptides of the formula:

CH₃-O-(CH₂-CH₂-O)_n-CH₂-CH₂-O-CH₂-CH₂-CH₂-NHT20 (III)

where n is 10-1,000 and NHT20 is a T20 polypeptide covalently linked through a terminal alpha-amino group. In one embodiment n is about 225, e.g., 227.

Such pegylated T20 polypeptides may be produced in any desired manner, preferably by the method described in example 3.

The pharmaceutical compositions of the present invention comprise a compound of formula (I) wherein R is in admixture with a pharmaceutically acceptable excipient₁M, n, p and NHT20 are as described above.

Pharmaceutical compositions of the invention comprising a pegylated T20 polypeptide or salt thereof can be produced in any desired manner, e.g., by 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. Excipients suitable for injection include water, alcohols, polyols, glycerol, 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, buffering agents, coating agents or antioxidants. They may also contain other therapeutically useful substances, including additional active ingredients.

Formulations suitable for parenteral administration (including subcutaneous, intramuscular, intravenous, intradermal, intratracheal and dural) may conveniently be presented in unit dosage form and may be prepared by conventional pharmaceutical techniques. Such techniques include the step of establishing a relationship between the pegylated T20 polypeptide and a pharmaceutical carrier or excipient. In general, formulations are prepared by thoroughly and intimately bringing into association the pegylated T20 polypeptide with a liquid carrier. Suitable formulations 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; aqueous and non-aqueous sterile suspensions which may contain 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) condition 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, or an appropriate fraction of the administered ingredients thereof, as described above.

The pegylated T20 polypeptide is preferably in unit dosage form. As used herein, "unit dosage form" means that the amount of pegylated T20 polypeptide suitable for a single dose is in a pre-weighed and/or pre-packaged form. This allows convenient preparation of pegylated T20 polypeptide for administration, even allowing self-administration by the patient. The amount of unit dose will obviously depend on the amount of pegylated T20 polypeptide to be delivered and the frequency of administration.

The pegylated T20 polypeptide can also be provided in the form of a lyophilized powder in a unit dosage amount that is suitable for reconstitution with a pharmaceutically acceptable excipient prior to administration.

Particular pharmaceutical compositions of the invention comprise a compound of formula (I) wherein R is in admixture with a pharmaceutically acceptable excipient₁Is methoxy, m is 1, n is 100-750, and p is 3.

Another pharmaceutical composition of the invention is a pharmaceutical composition comprising a compound of formula (III) wherein n is 10-1,000 and NHT20 is a T20 polypeptide covalently linked through a terminal alpha-amino group, in admixture with a pharmaceutically acceptable excipient. In one embodiment n is about 225, e.g., 227.

The invention also provides methods of inhibiting HIV infectionComprising administering to a patient a pharmaceutical composition comprising a compound of formula (I) wherein R is in admixture with a pharmaceutically acceptable excipient₁M, n, p and NHT20 are as described above.

Pegylated T20 polypeptides are typically administered as the current mode of administration of (non-pegylated) T20 polypeptides. However, some modifications may be made to take advantage of the improved pharmacokinetic properties of pegylated T20 polypeptides.

In the methods of the present invention for inhibiting HIV, the pharmaceutical composition may be administered in any suitable manner and route. In a preferred method, the pegylated T20 polypeptide is administered as an injectable solution or suspension. Preferably, the injectable solution or suspension is administered by subcutaneous injection or intravenous injection.

In another preferred method, the pegylated T20 polypeptide is administered via a transdermal delivery device, such as a transdermal patch.

In the methods of the present invention for inhibiting HIV, the pharmaceutical composition may be administered in any suitable dosage and regimen. The pharmaceutical compositions of the present invention may be administered in any desired form, by any desired route. However, the pegylated T20 polypeptides of the invention are typically administered parenterally, for example, in the form of an injection solution.

Determination of a therapeutically effective amount is within the skill of the art, and a therapeutically effective amount or dose of a pegylated T20 polypeptide according to the invention can be varied and adjusted as individually needed in each particular case. In general, in the case of parenteral administration to an adult human of about 70Kg in weight, a dosage of about 5 mg to about 300 mg per day should be suitable, preferably about 50 mg to about 200 mg, although the upper limit may be excessive as specified. This dose may be administered as a single dose, divided doses or as a continuous infusion. Administration may be daily, more preferably weekly.

The present invention also provides a method of inhibiting HIV infection comprising administering a pharmaceutical composition comprising a compound of formula (I) wherein R is in admixture with a pharmaceutically acceptable excipient₁Is methoxy, m is 1, n is 100-750, and p is 3.

Also contemplated within the scope of the present invention is a method of inhibiting HIV infection comprising administering a pharmaceutical composition comprising a compound of formula (III) wherein n is 10-1,000 and NHT20 is a T20 polypeptide covalently linked through a terminal alpha-amino group, in admixture with a pharmaceutically acceptable excipient. In one embodiment n is about 225, e.g., 227.

The following examples are provided to further illustrate the compounds, compositions, and methods of the present invention. These examples are intended to be illustrative only and are not intended to limit the scope of the present invention in any way.

Example 1

PEG_10KPreparation of butyraldehyde

mPEG (30.0 g, 3 mmol) with a molecular weight of 10,000 in 240 ml of toluene was azeotropically dried by refluxing for 2 hours, then 120 ml of toluene was removed. The resulting solution was cooled to room temperature and then a solution of potassium tert-butoxide (0.68 g, 6 mmol) in 20 ml of dry tert-butanol and 20 ml of toluene was added to the PEG solution. The resulting mixture was stirred at room temperature for two hours under argon. Tert-butyl bromoacetate (1.00 ml, 6.75 mmol) was added to the reaction via syringe and the reaction was stirred at room temperature under argon overnight. The reaction solution was then concentrated by rotary evaporation. Diethyl ether was added to precipitate the residue. Precipitated mPEG_10kThe tert-butylcarboxymethyl ester product was isolated by filtration and dried in vacuo. Yield: 28 grams. NMR (d)₆-DMSO)：1.40ppm(t，9H，-CH₃)；3.21ppm(s，-OCH₃)；3.50ppm(s，-O-CH₂CH₂-O-)；3.96ppm(s，2H，-O-CH₂-COO-)。

Then mPEG is added_10kTert-butylcarboxymethyl ester (26.5 g) was 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 adding 6N hydrochloric acid, and the mixture was then extracted with dichloromethane. The organic layer was dried over sodium sulfateDried, filtered, concentrated, and then precipitated into diethyl ether. Collection of m-PEG by filtration and vacuum drying_10k-a carboxymethyl acid product. Yield: 24 grams. NMR (d)₆-DMSO)：3.21ppm(s，-OCH₃)；3.5ppm(s，-O-CH₂CH₂-O-)；3.99ppm(s，2H，-O-CH₂-COOH)。

Then m-PEG_10k-carboxymethylacid (6 g, 0.6 mmol) was dissolved in anhydrous dichloromethane (30 ml) and 4-aminobutyraldehyde diethyl acetal (140 ml, 0.9 mmol), 1-hydroxybenzotriazole (80 mg, 0.6 mmol) and dicyclohexylcarbodiimide (160 mg, 0.78 mmol) were added. The mixture was stirred at room temperature under argon overnight. The reaction mixture was filtered, concentrated and precipitated with a mixture of 2-propanol and diethyl ether (1: 1). Mixing mPEG_10kThe butyraldehyde product was dried under vacuum overnight. Yield: 5.4 g. NMR (d)₆-DMSO)：1.07-1.12ppm(t，6H，(-O-CH₂-CH₃)₂)；1.46ppm(m，4H，-NHCH₂CH₂CH₂-CH-)；3.08-3.11ppm(q，2H，-NHCH₂CH₂CH₂-CH-)；3.21ppm(s，-OCH₃)；3.5ppm(s，-O-CH₂CH₂-O-)；3.85ppm(s，2H，-O-CH₂-CO-NH-)；4.44ppm(t，1H，-NHCH₂CH₂CH₂-CH-)；7.67ppm(-NH-)。

Then mPEG is added_10k-Butylal (2 g, 0.2 mmol) dissolved in 20 ml 80% CF₃In COOH, the solution was stirred at room temperature overnight. The pH of the mixture was adjusted to 6.0 by the addition of 1N NaOH solution, sodium chloride (10 wt%) was added and then 1N NaOH was added to adjust the pH of the solution to 7.0. The mixture was extracted with dichloromethane. The organic layer was dried over sodium sulfate, filtered, concentrated and precipitated into diethyl ether. Collection of mPEG by filtration and vacuum drying_10k-butyraldehyde product. Yield: 1.7 g. NMR (d)₆-DMSO)：3.21ppm(s，-OCH₃)；3.5ppm(s，-O-CH₂CH₂-O-)；3.85ppm(s，2H，-O-CH₂-CO-NH-)；7.67ppm(-NH-)；9.66ppm(-CHO-)。

Example 2

With PEG_10k-butyraldehyde PEGylated T20

To 3.0 ml of a buffer containing 15 mg of T20 (purity 93.7%) (50 mmol of potassium phosphate, pH6.5) was added butyraldehyde PEG (10kDa) in a molar ratio of 5 mol of reagent per one mol of T20 (see example 1). The T20 polypeptide is deacylated at the α -amino terminus, but protected at the carboxy terminus by-NH 2. To the reaction mixture was added 10% (V/V) of 0.5M aqueous solution of sodium cyanoborohydride and stirred at room temperature for 4 hours. The pegylated T20 was purified from the reaction mixture by ion exchange chromatography (QA). A linear gradient of increasing salt concentration, i.e., 150mM to 1M NaCl in 20mM Tris (pH 7.5), was used to separate pegylated T20 from unmodified T20.

Example 3

With mPEG_10KPropionaldehyde PEGylation T20

Propionaldehyde was esterified using PEG (10kDa) having the following structure

CH₃-O-(CH₂-CH₂-O)₂₂₇-CH₂-CH₂-O-CH₂-CH₂-CHO

To 3.0 ml of a buffer containing 15 mg of T20 (purity 93.7%) (50 mmol of potassium phosphate, pH6.5) was added 150 mg of mPEG at a molar ratio of 5 mol of reagent per one mol of T20_10k-propanal. The T1249 polypeptide is deacylated at the α -amino terminus, but protected at the carboxy terminus by-NH 2.

To the reaction mixture was added 10% (V/V) of a 0.5M aqueous solution of sodium cyanoborohydride and stirred at room temperature for 4 hours. The pegylated T20 was purified from the reaction mixture by ion exchange chromatography (QA). The pegylated T20 polypeptide has the structure:

CH₃-O-(CH₂-CH₂-O)_n-CH₂-CH₂-O-CH₂-CH₂-CH₂-NHT20 (III)

a linear concentration gradient of 150mM to 1M NaCl in 20mM Tris (pH 7.5) was used to separate pegylated T20 from unmodified T20.

Example 4

Inhibitor concentration of Pegylated T20

Phenotypic susceptibility is usually quantified by IC50 or IC90, i.e. the concentration of drug required to inhibit 50% or 90% of virus growth, respectively, is measured.

With PEG_10K-Pegylated T20 with propionaldehyde (example 3) and PEGylation with mPEG_10kIC50 and IC90 results for butyraldehyde pegylated T20 (example 2) are presented in table 1 below:

	IC50 (microgram/ml)	IC90 (microgram/ml)
			Pegylated T20 with PEG10K-propanal pegylated T20 (example 3)	0.261	3.074
With mPEG10k-butyraldehyde PEGylated T20 (example 2)	0.266	2.536

Table 1: inhibitor concentration 50 and inhibitor concentration 90 results.

IC50 and IC90 values were determined according to example 5.

Example 5

cMAGI/MAGI antiviral assay

These assays evaluate the reduction of infectious virus titer using the indicator cell line MAGI (multinuclear activation of galactosidase indicator) or the CCR 5-expression derived cell line cMAGI. The MAGI cell line was derived from parental HeLa cells by introducing the CD4 gene and the HIV-1 LTR-controlled b-galactosidase reporter gene with an amphotropic retroviral vector (Kimpton J, Emermann M, J Virol 66: 2232-9, 1992). The cMAGI cell line was derived from the MAGI cell line by introducing the CCR5 gene with the amphotropic retroviral vector PA317 (Chackerian B, LongEM, Luciw PA, Overbaugh J, J Virol 71: 3932-9, 1997). cMAGI cells support replication of the primary NSI (R5) isolate and the laboratory adapted strain X4 virus, whereas MAGI cells support replication of only the X4 virus. Both cell lines transactivated the expression of the b-galactosidase reporter gene under the control of the HIV-LTR by virtue of the ability of HIV-1 tat. The b-galactosidase reporter gene has been modified to localize to the nucleus and within a few days of infection can be detected in a strongly nuclear stained form with X-gal substrate. Thus, if there is only one infection cycle before staining, the number of stained nuclei can be considered equal to the number of infectious virions in the challenge inoculation.

Inhibitors of infection and cell-cell fusion, such as, for example, T20 or T1249(Wild C, Greenwell T, Matthews T, AIDS Res Hum Retroviruses 9: 1051-3, 1993), were added 24 hours after infection in order to allow confirmation that a cycle of infection was represented. Infected cells were counted using a CCD-imager, and both primary and laboratory adapted isolates showed a linear relationship between the number of viruses and the number of infected cells observed by the imager. In the MAGI and cMAGI assays, a 50% reduction in infectious titer (Vn/Vo ═ 0.5) was significant and provided the major cut-off value for evaluation of antiviral activity. The 90% reduction in infectious titer (Vn/Vo) was used as an additional cut-off to evaluate antiviral activity.

Dilutions of each test compound were tested in duplicate for resistance to the virus inoculum adjusted to approximately 1500-2000 infected cells/well in 48-well microtiter plates. The test compound is added to cMAGI or MAGI cells followed by the addition of the virus inoculum and 24 hours later inhibitors of infection and cell-cell fusion (Wild C, Greenwell T, Matthews T, AIDS Res Hum Retroviruses 9: 1051-3, 1993) are added to prevent secondary rounds of infection and intercellular viral spread. The cells were cultured for an additional 2 days, fixed, and stained with an X-galactosidase substrate to detect infected cells. The number of infected cells per control and test compound dilution was determined using a CCD-imager. IC50 was defined as the dilution of test compound that caused a 50% reduction in infectious viral titer. IC90 was defined as the dilution that caused a 90% reduction in infectious viral titer.

The invention being thus described, it will be obvious that the same may be 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.

Sequence listing

<110> Fuffman-Raroshi Co., Ltd

<120> Pegylated T20 polypeptide

<130>21328US

<150>US 60/398,195

<151>2002-07-24

<160>1

<170> PatentIn version 3.1

<210>1

<211>36

<212>PRT

<213> Artificial sequence

<220>

<223> peptide sequences of synthetic origin

<220>

<221>MOD_RES

<222>(36)..(36)

<223> residue 36 optionally modified with amino group

<400>1

Tyr Thr Ser Leu Ile His Ser Leu Ile Glu Glu Ser Gln Asn Gln Gln

1 5 10 15

Glu Lys Asn Glu Gln Glu Leu Leu Glu Leu Asp Lys Trp Ala Ser Leu

20 25 30

Trp Asn Trp Phe

35

Claims (14)

1. A compound of formula (I):

wherein

R₁Is a terminal group, and is a terminal group,

m is the number of 1 to 17,

n is 10 to 1,000,

p is 1-3, and

NHT20 is a T20 polypeptide covalently linked through its terminal alpha-amino group.

2. A compound according to claim 1, wherein R₁Selected from the group consisting of halogens, epoxides, maleimides, o-pyridines disulfide, tosylates, isocyanates, hydrazines hydrate, cyanuric halides, N-succinimidyloxy, thio-N-succinimidyloxy, 1-benzotriazolyloxy, 1-imidazolyloxy, p-nitrophenyloxy and

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

4. A compound according to claim 1, wherein p is 3, R₁Is methoxy, m is 1, n is 100-750.

5. A compound of the formula:

where n is 10-1,000 and NHT20 is a T20 polypeptide covalently linked through its terminal alpha-amino group.

6. A pharmaceutical composition comprising a compound of the formula:

wherein

R₁Is a terminal group, and is a terminal group,

m is a number from 1 to 17,

n is 10 to 1,000,

p is 1-3, and

NHT20 is a T20 polypeptide covalently linked through its terminal alpha-amino group.

7. Pharmaceutical combination according to claim 6Wherein p is 3, R₁Is methoxy, m is 1, n is 100-750.

8. The pharmaceutical composition according to claim 6, which is in the form of a freeze-dried powder.

9. The pharmaceutical composition according to claim 6, in the form of a solution or suspension for injection.

10. Use of a pharmaceutical composition comprising a compound of formula I in admixture with a pharmaceutically acceptable excipient for the manufacture of a medicament for inhibiting HIV infection:

wherein

R₁Is a terminal group, and is a terminal group,

m is a number from 1 to 17,

n is 10 to 1,000,

p is 1-3, and

NHT20 is a T20 polypeptide covalently linked through its terminal alpha-amino group.

11. The use according to claim 10, wherein the pharmaceutical composition is administered in an amount of from about 50 mg to about 200 mg per day.

12. The use according to claim 10, wherein the pharmaceutical composition is administered in a single weekly dose in an amount of from about 300 to about 1,500 mg.

13. Use of a pharmaceutical composition comprising a compound of formula I in admixture with a pharmaceutically acceptable excipient for the manufacture of a medicament for inhibiting HIV infection:

wherein

R₁Is a methoxyl group, and the compound is a methoxyl group,

m is 1, m is a linear chain,

n is 100-750 of the total weight of the alloy,

p is 3, and

NHT20 is a T20 polypeptide covalently linked through its terminal alpha-amino group.

14. A method of attaching a polyethylene glycol molecule to a T20 polypeptide, comprising reacting a T20 polypeptide with a polyethylene glycol aldehyde of the formula:

wherein

R₁Is a terminal group, and is a terminal group,

m is the number of 1 to 17,

n is 10 to 1,000, and

p is 1 to 3, and the compound has the structure of,

to produce a compound of the formula:

R₁-(CH₂CH₂O)_n-CH₂CH₂-O-(CH₂)_m-CO-NH-(CH₂)_p-CH₂-NHT20 (I)

wherein the polyethylene glycol aldehyde molecule is attached to the N-terminal amino group of the T20 polypeptide.