Patent Application: US-34753806-A

Abstract:
a method to reduce replication of hiv - 1 , involving administering a therapeutically effective amount of recombinant hnp4 to a subject in need thereof to combat hiv - 1 infection . the hnp4 agent may be utilized in pharmaceutical compositions including a pharmaceutically acceptable carrier and an anti - viral agent , e . g ., an anti - viral agent , or combination of such agents , such as nucleoside rt inhibitors , ccr5 inhibitors / antagonists , viral entry inhibitors , and functional analogs thereof .

Description:
the disclosures of the following references are hereby incorporated herein by reference : “ from pro defensins to defensins : synthesis and characterization of human neutrophil pro α - defensins - 1 and its mature domain ,” z . wu , et al ., journal of peptide research , volume 62 , issue 2 , page 53 - august 2003 ; “ productive folding of human neutrophil α - defensins in vitro without the pro - peptide ,” z . wu , et al ., j . am . chem . soc ., 125 ( 9 ), 2402 - 2403 , 2003 ; “ synthesis and characterization of human α - defensins 4 - 6 ,” z . wu , et al ., the journal of peptide research , volume 64 , issue 3 , page 118 - september 2004 ; and u . s . provisional patent application no . 60 / 649 , 873 filed feb . 3 , 2005 , in the names of w . lu , et al . for “ human neutrophil α - defensins 4 inhibits hiv - 1 in vitro .” the present invention provides a method to reduce replication of hiv - 1 , involving administration of a therapeutically effective amount of recombinant hnp4 . in one aspect , the invention relates to a pharmaceutical composition comprising hnp4 and a pharmaceutically acceptable carrier . in another aspect , the present invention contemplates a method of treating a subject to reduce replication of hiv - 1 in such subject , comprising administering to such subject an effective amount of hnp4 , as therapeutically effective to reduce replication of hiv - 1 in said subject , in relation to replication of hiv - 1 in such subject in the absence of such therapeutic intervention involving the hnp4 therapeutic agent . the subjects to be treated by such method include human subjects . depending on the specific level of hiv - 1 infection of such subject , the subject may be administered hnp4 in the pharmaceutical composition , at any suitable therapeutically effective and safe dosage , as may readily determined within the skill of the art , and without undue experimentation . in general , while the effective dosage of the hnp4 therapeutic agent may be widely varied in the broad practice of the invention , depending on the specific level of infection of hiv - 1 in the subject , as readily determinable within the skill of the art , suitable therapeutic doses of hnp4 for achievement of therapeutic benefit may in various specific instances be in a range of 1 microgram ( μg ) to 100 milligrams ( mg ) per kilogram of the subject per day , e . g . in a range of 5 μg to 75 mg per kilogram body weight per day , or alternatively in another embodiment in a range of 10 μg to 75 mg per kilogram body weight per day , or alternatively in a further embodiment in a range of 10 μg to 50 mg per kilogram body weight per day . such dose may be presented as a single dose or two or more sub - doses administered at the appropriate intervals , and the sub - doses may be administered in unit dosage forms , for example , containing from 10 μg to 1 , 000 mg , such as from 50 μg to 500 mg , or 50 μg to 250 mg , or from 50 μg to 10 mg per unit dosage form , in various embodiments . alternatively , if the condition of the subject so requires , the doses may be administered as an infusion . the mode of administration and dosage forms will of course affect the therapeutic amount of the hnp4 therapeutic agent that is desirable and efficacious for the given treatment application . the administrative methods include any suitable methods such as parenteral administration , oral administration , intrathecal administration , pulmonary administration , etc ., as appropriate to a specific therapeutic regimen . specific administration modalities include : oral , rectal , topical , nasal , ophthalmic , subcutaneous , intramuscular , intravenous , transdermal , spinal , intra - articular , intra - arterial , sub - arachnoid , sub - lingual , oral mucosal , bronchial , lymphatic and intra - uterine . depending on the administration modality , the pharmaceutical composition may be provided in liquid solution or suspension form , or as a powered solid , for administration . in some applications it may be advantageous to utilize the hnp4 in a “ vectorized ” form , such as by encapsulation in a liposome or other encapulsant medium , or by fixation of the active agent , e . g . by covalent bonding , chelation , or associative coordination , on a suitable biomolecule . the pharmaceutical composition may therefore be formulated in any suitable manner , within the skill of the art . in one embodiment , the invention contemplates a pharmaceutical composition comprising recombinant hnp4 , a pharmaceutically acceptable carrier and an anti - viral agent . the anti - viral agent may be of any suitable type , as described more fully hereinafter . in another composition , the invention contemplates a formulation including hnp4 and a pharmaceutically acceptable carrier , in which the hnp4 comprises amino acid residues 64 - 96 of defensin 4 . in the pharmaceutical compositions including hnp4 , the pharmaceutically acceptable carrier may include any suitable carrier components or formulations , which are pharmaceutically acceptable in the sense of being compatible with the other ingredients of the formulations and not unduly deleterious to the subject . in another aspect , the invention contemplates a method for folding hpns , comprising generating an amino acid sequence of hpn lacking a pro - region , and folding such sequence in the presence of urea and n , n - dimethylformamide ( dmf ). inhibition of hiv - 1 in vitro by α - defensins 1 - 3 was first reported by nakashima et al , 1993 [ 11 ], which has been confirmed by several recent reports [ 4 , 12 ]. to demonstrate that synthetic hnps are active against hiv - 1 infection in vitro , we tested hnps for their inhibition of two laboratory isolates hiv iiib ( x4 ) and hiv bal ( r5 ) and two primary isolates b703 ( x4 ) and nsi03 ( r5 ) in pbmc using our standard assay protocols . human neutrophil α - defensins ( hnps ) are small , cys - rich , cationic antimicrobial proteins . stored in the azurophilic granules of neutrophils , they are released during phagocytosis to kill ingested foreign microbes through disruption of their cytoplasmic membranes . hnps are synthesized as inactive precursors in vivo and activated through proteolytic removal of their inhibitory n - terminal pro - peptide required for correct subcellular sorting and processing . folding of hnps in vitro without the pro - peptide has been reported to be extremely difficult which led to the hypothesis that the 45 - residue anionic pro - peptide may assist prohnps folding as an intramolecular chaperone interacting with the cationic c - terminal domain , a mechanism reminiscent of some bacterial serine proteases . however , we have shown that hnps without the pro - region can fold productively with yields over 80 % in the presence of 2 m urea and 25 % n , n - dimethylformamide ( dmf ). our finding demonstrates an efficient protocol for the production of large quantities of highly pure human α - defensins and is broadly applicable in folding aggregation - prone , cys - rich proteins of both synthetic and recombinant origin . human α - defensin 4 , i . e ., hnp4 , can be synthesized as described in “ synthesis and characterization of human α - defensins 4 - 6 ,” z . wu , the journal of peptide research , volume 64 , issue 3 , page 118 - september 2004 , the description of which is incorporated herein in its entirety for all purposes . a method for generating properly folded hnp4 is described in zhibin wu , robert powell , and wuyuan lu am . chem . soc ., 125 ( 9 ), 2402 - 2403 , ( 2003 ), “ productive folding of human neutrophil α - defensins in vitro without the pro - peptide ” the description of which hereby is incorporated by reference herein for all purposes . folded hnps are known to adopt a three - stranded anti - parallel β sheet conformation constrained by three intramolecular disulfide bonds . they are highly soluble in aqueous solution and are structurally stable even in 8 m urea . in contrast , the reduced form quantitatively aggregates in the absence of high concentrations of denaturants . consequently , massive precipitation occurs as denaturing conditions are removed during the folding of hnps in aqueous solution . currently , the most effective protocol for folding hnps without the pro - peptide utilizes the irreversible and nondiscriminating oxidant dmso and typically results in 10 % recovery . due to the exceptionally high stability of hnps , the prevention of aggregation of unfolded proteins , by using sufficient amounts of denaturants , suitable organic cosolvents , or both , may facilitate productive thiol - disulfide exchanges in the presence of oxidized / reduced thiol pairs , thus leading to the formation of a stable native structure . nonetheless , we do not wish to be bound by any theory or mechanism , as regards the specific basis or reasons for such exceptionally high stability . cells infected with the hiv - 1 virus were added to duplicate series of wells containing varying concentrations of test defensin in 200 ul complete culture medium , and fed after 48 hours with fresh defensin of appropriate quantities . medium alone was used as the control . the level of hiv - 1 replication was measured at day 4 after infection by hiv - 1 p24 antigen - capture elisa . percent infection was determined as a function of hiv p24 released into the medium in sample wells versus in control wells . the data are shown in fig1 - 4 . fig1 is a graph of percentage inhibition of hiv - 1 as a function of defensin concentration , μm for hiv - 1 isolate iiib ( x4 ) in pbmc by hnps 1 - 4 , wherein -∘- is hnp1 , - δ - is hnp2 , - x - is hnp3 and -•- is hnp4 . fig2 is a graph of percentage inhibition of hiv - 1 as a function of defensin concentration , μm for hiv - 1 isolate bal ( r5 ) in pbmc by hnps 1 - 4 , wherein -∘- is hnp1 , - δ - is hnp2 , - x - is hnp3 and -•- is hnp4 . fig3 is a graph of percentage inhibition of hiv - 1 as a function of defensin concentration , μm for hiv - 1 isolate b703 ( x4 ) in pbmc by hnps 1 - 4 , wherein -∘- is hnp1 , - δ - is hnp2 , - x - is hnp3 and -•- is hnp4 . fig4 is a graph of percentage inhibition of hiv - 1 as a function of defensin concentration , μm for hiv - 1 isolate nsi03 ( r5 ) in pbmc by hnps 1 - 4 , wherein -∘- is hnp1 , - δ - is hnp2 , - x - is hnp3 and -•- is hnp4 . all four α - defensins inhibit their susceptible hiv - 1 strains in a dose - dependent manner , with similar ic 50 values for hnps 1 - 3 in the range of 5 - 20 um . however , inhibition of both strains of hiv - 1 by hnp4 is significantly and unexpectedly stronger as compared with that achieved by hnps 1 - 3 . 1 . lehrer , r . i ., and ganz , t . 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