Patent Application: US-94014707-A

Abstract:
residual hiv - 1 replication reemerges after intensive therapy from location or locations in the body called the drug resistant non - plasma viral reservoir . methods are disclosed of treating hiv by inhibiting or blocking this reemergence with various monomeric therapeutic peptide compositions including monomeric dapta prepared in least 80 % trifluoroethanol , with vigorous shaking for at least about 24 hours at about 37 ° c .

Description:
the present invention may be more readily understood by reference to the following specific embodiments and examples a method for reducing or preventing the aggregation of a therapeutically active peptide , polypeptide or variant thereof upon reconstitution by admixing a therapeutically active peptide , polypeptide or variant thereof and a fluorinated organic solvent , removing the fluorinated organic solvent , and reconstituting the therapeutically active peptide , polypeptide or variant thereof with an aqueous solution , thereby increasing the biological activity of the therapeutically active peptide , polypeptide or variant thereof by eliminating minor contaminant aggregates which form seeds that then rapidly promote further aggregation leading to fibril formation and loss of bioactivity . the fluorinated organic solvents include but are not limited to trifluoroethanol ( tfe ) and 1 , 1 , 1 , 3 , 3 , 3 ,- hexafluoro - 2 - propanol ( hfip ). concentrations of fluorinated organic solvents , where tfe is a favored embodiment , comprise 80 to 100 %. although use of the lower tfe concentration is possible , use of 100 % is favored as it avoids an artifact introduced by drying which would partition some of the peptide into the residual water phase at the high concentrations which promote aggregation . concentrations of peptide should be maintained at 5 mg / ml or less in the final reconstitution in water , in the absence of salts , especially nacl , which is commonly used to prepare physiological solutions . the invention also provides a method for increasing the biological activity , promoting storage stability , and preventing or eliminating aggragation of a therapeutically active peptide , polypeptide or variant thereof by reconstitution in a fluorinated organic solvent with vigorous shaking at warm temperatures , from 37 to 56 c for 24 hours or longer . the invention also provides a method for increasing the biological activity of a therapeutically active peptide , polypeptide or variant thereof upon reconstitution by admixing a therapeutically active peptide , polypeptide or variant thereof and a fluorinated organic solvent , shaking in the warm , removing the fluorinated organic solvent , and reconstituting the therapeutically active peptide , polypeptide or variant thereof with an aqueous solution , thereby increasing the biological activity of the therapeutically active peptide , polypeptide or variant thereof . still , the invention provides a method for reducing or preventing fibrillar formation of a therapeutically active peptide , polypeptide or variant thereof upon reconstitution by admixing a therapeutically active peptide , polypeptide or variant thereof and a fluorinated organic solvent , removing the fluorinated organic solvent , and reconstituting the therapeutically active peptide , polypeptide or variant thereof with an aqueous solution , thereby increasing the biological activity of the therapeutically active peptide , polypeptide or variant thereof . the therapeutically active peptide , polypeptide or variant thereof of the invention includes but is not limited to peptide t or an analog thereof , including d - ala 1 - peptide t - amide , d - ala 1 - peptide t lacking an amide at the c - terminus , d - ala 1 thr 8 - peptide t amide , vasoactive intestinal peptide ( vip ), thr - thr - ser - tyr - thr ( seq . id no . : 1 ). peptides in the normal l form , may be prepared and used in this manner . dapta was prepared by the methods described herein to be free of aggregates . these aggregated peptide forms are themselves biologically inactive due to their inability to bind to ccr5 , and their presence in minute amounts in stored solutions promotes further aggregation and fibril formation , with concomitant loss of biological activity and clinical effect . standard and typical formulations used in previous trials ( 21 ), or laboratory studies ( 23 ) have lost activity , or been devoid of activity . the reasons for variable and negative results have been confounding and until now unknown , with pernicious and deleterious effects for meeting treatment needs in hiv / aids . viral replication and production in hiv - infected m / m treated with dapta was assessed 14 and 21 days after infection for p24 antigen production . a representative experiment is shown in fig1 . the ccr5 - using hiv - 1 strain , 81a had p24 gag antigen production of control m / m cultures reduced ˜ 80 % by doses of monmeric dapta as low as 10 − 14 m ( fig1 ). dapta had no effect on hiv - 1 p24 antigen production in m / m infected by cxcr4 - using ( x4 ) strains , hiv - 1 iiib ( data not shown ). these results are some 100 - 100 , 000 fold lower than described in earlier reports . thus sodroski failed to find antiviral effects of dapta at doses as high as 10 − 7 m ( 23 ), and effective in vitro doses were typically reported to be 10 − 12 m to 10 − 9 m ( 17 ). thus this improved formulation enhanced in vitro potency by 100 to 100 . 000 - fold . preparation and storage of dapta , without modifications to prevent aggregation , can rapidly result in orders of magnitude loss of potency , to confirm that monomeric dapta binding is specific for ccr5 , a competition experiment between ccr5 - fitc antibody and dapta in m / m was done . flow cytometric analysis showed that 20 % of dapta treated m / m are ccr5 + positive . treatment with monomeric dapta reduced ccr % expression to 9 % with 10 − 12 m dapta , compared with 35 % of mock - treated m / m ( fig2 ) ( p ≦ 0 . 001 ). overall , the inhibition of ccr5 - binding by several dapta doses is about 43 % and reaches a maximum of 73 % with 10 − 12 m . these results clearly indicate that dapta reduced the ccr5 antibody binding to the receptor in m / m by down - regulating receptor expression . dapta compositions which did not have reduced or absent aggregates failed to down - regulate ccr5 expression ( data not shown ). monomeric dapta reduces levels of hiv - 1 dna in human primary macrophages to further prove that monomeric peptide dapta blocks virus infection with increased potency , m / m were analyzed for hiv - 1 dna formation . eighteen hours postinfection , genomic dna was extracted and two - fold dilution of cell equivalents ( range 1 × 10 6 - 1 . 25 × 10 5 ) were amplified in an inverse / nested pcr specific for a conserved gag region of the viral genome . semi - quantitative analyses of hiv - 1 dna in m / m were performed by comparison of dna amplification products from infected cells , standardized by pcr for β - actin , to standards of amplified u1 dna copies and cell numbers . the un - scan it - gel software ( silk scientific inc .) was used to determine band densities ( fig3 a ). we observed that hiv - 1 dna per 2 . 5 × 10 5 cells declined with 64 % in the presence of peptide dapta ( 10 − 7 m ) and with 70 % in the presence of 10 − 9 m peptide dapta , compared with not - treated cells . in the absence of peptide dapta or 2d7 mab , approximately 1 × 10 4 hiv - 1 copies were presented per 10 5 m / m ( i . e . 0 . 1 copy × m / m ). the inhibition of hiv - 1 dna formation detected in m / m in the presence of mab 2d7 at the maximum amount of 3 μg / ml was approximately 39 % ( fig3 b ). in conclusions , these data indicate that monomeric dapta inhibits productive infection in m / m by blocking specifically the ccr5 dependent entry with a potency greater than that of the specific anti - ccr5 antibody 2d7 . the results suggest use as a therapy for treatment resistant monocyte / macrophage or t cell infection in hiv / aids . monomeric dapta effects on ccr5 binding and gp120 - induced apoptosis in neuronal cell lines to assess ccr5 expression on surface of neuronal cell lines , sk - n - sh were stained with 2d7 mab in presence or in absence of dapta ( at different doses ) and tak - 779 . sk - n - sh line has the potential of differentiating to neural cells in the presence of retinoic acid , and it has been used as a model of primary neurons . the results indicate that ccr5 expression in these differentiated cells is limited and further reduced in the presence of dapta ; indeed an inhibition of ccr5 expression of 68 . 5 % and 72 % in presence of 10 − 13 m and 10 − 12 m dapta concentration respectively was observed in comparison with unexposed sk - n - sh ( fig4 ) ( p & lt ; 0 . 001 ). in the presence of tak - 779 ( 1 . 8 × 10 − 6 m ) the inhibition is about 61 %. thus , monomeric dapta is considerably more potent than tak - 779 to downmodulate ccr5 coreceptor expression in a neuronal cell line . finally , we exposed differentiated sk - n - sh cells to the r5 hiv - 1 strain bal , in the presence or absence of monomeric dapta , and assessed neuronal apoptosis . time - course studies revealed that cell apoptosis in this cellular line occurred between 5 and 6 days after addition of the virus . thus results will be shown at day 5 . in particular , when sk - n - sh were incubated with hiv - 1 bal , a dramatic reduction of cell viability was seen by facs analysis . the cytopathic effect , observed in sk - n - sh exposed to r5 hiv - 1 released from infected m / m , was mainly related to apoptosis . indeed , facs analysis showed apoptosis in 60 % of sk - n - sh cells exposed to hiv - 1 bal compared to 28 % and 26 % observed in dapta 10 − 3 m and 10 − 12 m treated cells , respectively . to compare the anti - apoptotic effect of monomeric dapta with other ccr5 - binding molecules , we also tested the ccr5 antagonist tak - 779 . sk - n - sh cells treated with 1 . 8 × 10 − 6 m tak - 779 ( a concentration able to strongly inhibit virus replication in m / m ) resulted in only a 30 % inhibition of apoptosis compared with the cells not treated with tak - 779 ( fig5 ). these data also indicated that monomeric dapta is more potent in preventing the neuronal apoptosis compared to tak - 779 and has increased potency compared to non - monomeric peptide preparations . in treating hiv infection , useful peptide concentrations in the composition can range from about 5 mg / ml to about 0 . 00005 mg / ml . effective blood plasma levels are expected to range from about 10 − 9 m to about 10 − 17 m . the inventors specifically contemplate the use of all concentrations within these ranges depending on the surrounding circumstances . for example , treated patients can be in different stages of infection , have different genetic or physiologic backgrounds . different concentration levels will be optimal as one balances drug potency and adverse side effects . 1 . blankson , j . n ., d . persaud , and r . f . siliciano . 2002 . the challenge of viral reservoirs in hiv - 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