Patent Application: US-28596308-A

Abstract:
we disclose here a method for using poly - iclc to prevent and / or treat certain human and veterinary infectious , neoplastic and autoimmune disorders , as well as for regulating a broad variety of genes in humans , consisting of use of poly - iclc repeatedly and at low doses , alone or in combination with other drugs or vaccines . as such it represents an example of broad spectrum host - targeted therapeutics , in contrast to conventional antibiotics , antiviral or antineoplastic agents that target specific organisms or tumors .

Description:
the invention is a novel method for clinical use of poly - iclc with reduced toxicity at effective dose levels , and method for using poly - iclc to regulate genes and treat certain disease conditions . the complex interaction of the interferons and dsrna - activated systems can be manipulated to therapeutic advantage , particularly in the case of those certain microbial and neoplastic diseases that thrive by circumventing host defense mechanisms such as those involving the oas , pkr , rig - i , mda5 , and tlr . one approach described by marcus and colleagues in avian cell culture uses the combination of exogenous interferon followed by dsrna in that order to achieve an up to 100 - fold increased level of protection against avian reovirus and newcastle disease virus over that provided by either agent alone or when given in the reverse order ( marcus and sekellick 2001 ). a similar rationale can be used to treat certain human and veterinary infectious and neoplastic diseases utilizing poly - iclc alone . in this context , poly - iclc is serving two functions ; first the induction of interferon and gene expression for oas , pkr , rig - i tlr3 and certain other proteins , and second the activation of these previously induced dsrna dependent systems such as oas , pkr , and other enzymes . this approach better mimics the repeated dsrna presentation to the host that results from most viral replication , but that is missing or is inhibited by evasive mechanisms in most cancers , viral infections , or with most vaccines . the approach described here is to first stimulate interferon induction with a moderate to high dose of poly - iclc , allow 4 - 72 hours for interferon induction of oas , pkr and other genes , and then activate them with a second , lower dose of poly - iclc . given the short ( 2 - 3 day ) half - life of these systems , this cycle may need to be repeated at least once or twice a week for a variable period of time depending on the disease in question . in some infections , such as mouse influenza , a single two - dose cycle of intranasal poly - iclc ( 1 mg / kg ) has been shown to protect from lethal viral challenge for as long as two weeks , although the authors did not specifically pursue the high dose - low dose sequence disclosed here ( wong , saravolac et al . 1995 ) a single dose cycle was less effective . likewise , in the treatment of malignant gliomas , we found that less than twice weekly poly - iclc dosing appeared ineffectual . ( salazar , levy et al . 1996 ) in that successful clinical trial ( further described in the example below ), dosing was spaced at 48 hours . thus the second dose of poly - iclc was given at a time when the oas ( and presumably pkr ) had reached peak blood levels from the first dose and could be most effectively activated by the poly - iclc . clinical trials of poly - iclc to date have utilized the intravenous ( iv ) or intramuscular ( im ) routes of administration . we have demonstrated that intranasal poly - iclc can protect from otherwise lethal nasal sars coronavirus challenge in mice ( barnard , salazar , et al , unpublished , see fig3 . we have now have demonstrated that intranasal treatment of non - human primates ( rhesus monkeys ) with poly - iclc will also result in a robust systemic response as measured by plasma interferon at 24 hours , but not 8 hours from administration , and as shown in fig1 . this unexpected result opens the possibility of using intranasal , sublingual , or topical poly - iclc for treatment of systemic diseases . this may be especially advantageous in long term treatment of cancer or autoimmune diseases , treatment of large number of individuals exposed to a bioterror threat such as smallpox ; or for veterinary use , as in containment of an outbreak of foot and mouth disease in cattle , management of the bovine respiratory complex , avian influenza , or the porcine reproductive - respiratory syndrome ( prrs ) similarly , it is also expected that orally or sublingually administered poly - iclc may be sufficient to yield a clinical therapeutic response . oral administration could also be especially advantageous in large - scale human or veterinary uses . finally , older rabbit studies demonstrated protection from vaccinia by topically administered poly - iclc . this suggests that topical administration of poly - iclc in a dermatologic preparation or dermal patch may also be efficacious for certain applications in humans ( levy and lvovsky 1978 ). further data will be presented to address these claims . an improved method for the clinical and veterinary use of poly - iclc poly - iclc , especially as improved as described above , has application to the treatment of a variety of diseases including certain neoplastic , infectious , and autoimmune disorders . the following examples are illustrations , but not limitations , of the approach . given these examples , one of ordinary skill in the art could apply the same approach to other diseases . a ) example of clinical gene modulation by poly - iclc in primates , including man human patients with recurrent malignant gliomas undergoing therapy with poly - iclc ( 20 mcg / kg im 3 times per week ) had blood drawn at baseline and 24 hours after the first injection , white cells were isolated and frozen . rtpcr was used to evaluate the expression of the gene for interferon inducible p56 enzyme in wbc . the figure shows up to a several hundred - fold increase in gene expression at 24 hours . ( sen g , salazar a m , et al , unpublished ) ( see fig1 ) additional studies will further demonstrate the spectrum of gene activation , including but not limited to the following genes : helicase , and , tumor necrosis factor , interferon regulatory factor , matrix metalloproteinase , plasminogen activator , tumor protein p53 , fibroblast growth factor , eukaryotic initiation factor 2 , actin filament - associated protein , and vcam - 1 . these studies will not only demonstrate the spectrum of gene activation in humans by low dose poly - iclc , but will also reveal possible correlations to tumor response . nevertheless , the potential clinical therapeutic uses of the ability to regulate such a broad variety of genes extends beyond the infection and neoplasm treatments described below . poly - iclc ( 10 to 50 mcg / kg intramuscularly one to three times weekly ) was given for up to 56 months to 38 patients with glioblastoma multiforme or anaplastic astrocytoma . ( salazar , levy et al . 1996 ) there was relatively low or no toxicity . twenty of 30 patients ( 66 %) receiving at least twice weekly poly - iclc ( including all anaplastic astrocytoma patients ) showed regression or stabilization of enhancing tumor on mri ( median = 65 % volume decrease ). only two of the 11 anaplastic astrocytoma patients subsequently showed tumor recurrence while on poly - iclc , and most of the group remain alive , with a median progression - free follow - up of over 65 years from diagnosis ( range 22 - 134 + months ). median overall survival is now 111 + months ( range 34 - 134 +). median kaplan - meir survival for glioblastoma patients on at least twice weekly poly - iclc treatments was 19 months ; only one remains alive ( 98 months from diagnosis ). tumor response was associated with 2 ′ 5 ′ oligoadenylate synthetase activation ( p = 0 . 03 ), but not with serum interferon , interleukins , or neopterin . the 100 % sustained tumor response or stable rate , and the prolonged , continuing , quality survival in anaplastic astrocytoma patients on poly - iclc contrasts favorably with the expected median survival of about 26 months for newly diagnosed aa patients on traditional chemotherapy . as suggested above , it is expected that even better survivals than those seen to date may be achievable utilizing a new double - dosing technique , as follows : poly - iclc is administered intranasally , orally , sublingually , intramuscularly , intravenously or topically in at least two doses spaced 4 - 72 hours apart . preferably , the first dose is in a moderate range sufficient to induce measurable but not excessive levels of serum interferon ( 30 to 100 mcg / kg in humans ); and the second , lower dose is in the maximally effective range for unblocking and stimulation of certain interferon and dsrna inducible enzyme systems , including the pkr and 2 ′ 5 ′ oas , which reach their serum peaks some 48 hours post initial poly - iclc dosing . for humans , the first dose would preferably be in the range of 30 to 100 mcg / kg and the second dose would preferably be in the range of 10 to 40 mcg / kg . preferably , the doses would be spaced approximately 48 hours apart . ( see updated , unpublished survival data in table in fig2 ) it is expected that this could be confirmed or suitably modified by those skilled in the art , based on the outcome of ongoing phase ii clinical trials of poly - iclc for patients with malignant brain tumors . a number of cancers share various characteristics with malignant gliomas , and likely utilize similar mechanisms to avoid host defenses . such cancers may thus also be amenable to poly - iclc treatment using the regimen described herein . they include melanoma , and certain leukemias and lymphomas , which share abnormalities on chromosome 9p ; renal cell carcinoma ; and sarcomas , lung , breast , and colon cancers that occur together with gliomas in the familial li - fraumeni syndrome . c . example of use of nasal poly - iclc for prevention of sars cov infection . sars (‘ severe acute respiratory syndrome ), is a classical example of the danger posed by emerging pathogens . it appeared in asia in 2003 and rapidly became a global outbreak , even before the causative agent , a rare coronavirus , was identified . there was no effective treatment and of 8 , 000 cases worldwide , nearly 800 died . beyond the human loss , the economic disruption was considerable . we have now demonstrated complete protection by nasally administered [ poly - iclc ] in an otherwise lethal murine challenge models of sars . [ poly - iclc ] appears to be the most effective such treatment tested to date and has become the ‘ treatment control ’ or ‘ gold standard ’ of therapy in this model . mice were treated with 0 . 25 to 1 mcg / kg of nasal poly - iclc prior to or after challenge with a lethal dose of sars coronavirus . primary outcome was survival , as indicated . pretreatment with even 0 . 25 mg / kg poly - iclc in repeated doses at 24 hour intervals provided total protection from an otherwise lethal viral challenge . ( barnard d , salazar a m , et al , unpublished ) see fig3 . these studies are further evidence of the potential utility of nasal poly - iclc for providing immediate broad spectrum protection and epidemic containment in the case of an intentional ( bioterror ) or unintentional outbreak of infections with emerging respiratory pathogens . treatment of aids with poly - iclc . in an open pilot trial , low dose ( 0 . 2 - 2 mg ) piclc was administered intramuscularly ( im ) 1 - 3 times per week with or without zidovudine over up to 30 months to 22 patients with hiv infection or aids . ( salazar , morales et al . 1990 ) piclc was well tolerated , with no significant clinical or laboratory toxicity . side effects consisted of a mild 12 - 24 hour flu - like syndrome with low - grade fever and malaise at the higher doses , but usually disappeared after the first half - dozen treatments . 12 / 20 patients showed at times dramatic initial rises in t - 4 cell counts along with symptomatic improvement , although this was not uniformly maintained . plasma p - 24 titers ( a measure of viral load ), which were positive in 8 / 16 patients before biweekly treatment , either became undetectable or remained so in all but one patient , whose titers were markedly elevated at onset and dropped by 75 % with treatment . in a separate dosing study of piclc in 8 aids patients , neuropyschological testing has shown a marked improvement in choice reaction time and the purdue pegboard test at 16 weeks of treatment , with a deterioration back to baseline when piclc was discontinued . ( salazar , martin , unpublished ). this contrasts with a gradual , statistically significant deterioration in choice reaction time seen in an untreated hiv + cohort ( n = 41 ) over six months . as suggested above , it is expected that even better responses than those seen to date may be achievable utilizing the new double - dosing technique described in section b , above , and shown in fig4 . an open trial of high dose ( 100 mcg / kg ) intravenous piclc showed moderate acute toxicity in 15 patients with chronic ms ; several patients improved or stabilized , but deteriorated when drug was stopped , as reported by bever , salazar , et al ., 1986 . ( bever , salazar et al . 1986 ) subsequently , salazar continued to treat some of these and other ms patients with a completely new extended regimen using much lower doses of piclc intramuscularly over a longer period of time . results of this follow - up study are unpublished , and are disclosed below and in fig5 methods : thirty one patients with either chronic progressive ( cp ) or exacerbating progressive ( ep ) multiple sclerosis received 5 - 100 mcgm / kg piclc im q 3 - 14 days for up to 15 years ; most received a median dose of 10 mcg / kg weekly . toxicity was markedly reduced to an inconstant , mild , transient malaise . the kurtzke expanded disability status score ( edss , which varies between 0 ( normal ) and 9 ( totally bedridden and dependent ) was used to evaluate outcome . as shown in fig6 , the edss remained stable or improved in 15 / 31 patients ( dramatically in 5 ). six patients deteriorated when piclc was stopped . the 19 cp patients showed a median edss change of 0 . 09 points per year over a median of 60 months ; while the 12 ep patients showed a slight improvement (− 0 . 1 edss per year ) over a median of 28 months . these rates compare very favorably with the expected rates of progression in untreated multiple sclerosis patients . ( note that a lower kurtzke score is better ). therefore , im low dose piclc may be a valuable alternative to the more expensive and toxic beta - interferons for long - term management of ms . as suggested above , it is expected that even better response rates than those seen to date may be achievable utilizing the new double - dosing technique described above . iv . a clinical method for increasing the speed and efficacy and decreasing the toxicity of vaccines .). the strategy presented here is designed to simultaneously control vaccine side effects by decreasing viral proliferation while at the same time targeting the relevant antigens for an immune response to vaccination . it does so by stimulating the host &# 39 ; s natural immediate response mechanisms to viral infection , including local dendritic cell activation at the time of vaccination through im or topical administration of poly - iclc at the time of vaccination . while illustrated with respect to a to method for producing large lots of final sterile poly - iclc suitable for clinical use with reduced toxicity at effective dose levels , and method for using poly - iclc to regulate genes , and manage certain disease conditions , the invention may be applied to any method for producing large lots of final sterile poly - iclc suitable for clinical use with reduced toxicity at effective dose levels , and method for using poly - iclc to regulate genes using the same techniques , modified to adapt to method for producing large lots of final sterile poly - iclc suitable for clinical use with reduced toxicity at effective dose levels , and method for using poly - iclc to regulate certain genes in a manner which would be known to one skilled in the art . baron , s ., a . salazar , et al . 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