The invention relates to viral formulations and related pharmaceutical products for use in gene therapy and/or vaccine applications. Especially preferred viral formulations disclosed herein are liquid adenovirus formulations, which show improved stability when stored in about the 2-8.degree. C. range while also being compatible with parenteral administration. These formulations comprise a buffer, a sugar, a salt, a divalent cation, a non-ionic detergent, as well as a free radical scavenger and/or a chelating agent to inhibit free radical oxidation.

Description of the Invention

SUMMARY OF THE INVENTION

The present invention relates to stabilized virus formulations and related pharmaceutical products for use in gene therapy and/or vaccine applications. A preferred viral formulation, as disclosed herein, may related to liquid formulations which comprise a recombinant adenovirus, formulations which show improved viral stability when stored in about the 2-8.degree. C. range and higher while also being compatible with parenteral administration. These formulations may comprise a buffer, a sugar, a salt, a divalent cation, a non-ionic detergent, as well as additional components which enhance stability of the included virus, including but not limited to a free radical scavenger and/or a chelating agent. The adenoviral-based formulations of the present invention are amenable to prolonged storage at 2.degree. C. to 8.degree. C. and higher for periods approaching two years. The recombinant viruses of the present invention which show enhanced storage stability include but are not limited to adenovirus, adeno-associated virus, retroviruses, herpes virus, vaccinia virus, rotovirus, pox viruses. The preferred virus is an adenovirus, including but not limited to human Ad5, Ad2, Ad6, Ad24 serotypes, and especially recombinant adenoviral virus for use in human gene therapy or human gene-based vaccination technology, including a prophylactic or therapeutic application utilizing such a gene-based vaccination technology.

The formulations of the present invention are (i) optimally a buffered solution and further comprise (ii) a minimal amount of at least one non-ionic surfactant; (iii) a divalent cation; (iv) a cryoprotectant; (v) a salt, and (vi), preferably inclusion of one or more additional excipients that act as inhibitors of free radical oxidation. The formulations of the present invention rely on a useful range of total osmolarity which promotes long term stability at temperatures of 2-8.degree. C., or higher, while also making the formulation useful for parenteral, and especially intramuscular, injection.

To this end, a first embodiment of the present invention relates to a series of adenovirus formulations (including but not limited to a recombinant adenovirus) which comprise Tris as the buffer, sucrose as the cryoprotectant, NaCl as the salt, MgCl.sub.2 as the divalent cation and either Polysorbate-80 or Polysorbate-40 as the surfactant.

A second embodiment of the present invention relates to inclusion of one or more inhibitors of free radical oxidation, including both metal ion chelators and hydroxyl radical scavengers, which are shown herein to enhance short and long term stability of the virus formulations described herein (again, including but not limited to an adenovirus, including a recombinant adenovirus containing a transgene, or portion thereof, which is useful in gene therapy and/or gene vaccination technology). Therefore, a preferred embodiment of the present invention is a viral formulation which contains one or more components which act as an inhibitor of free radical oxidation. It is shown herein that addition of these components enhance long term stability at temperatures up through the 2-8.degree. C. range, or higher, when compared to core formulations which do not contains these inhibitors. These formulations are also compatible with parenteral administration. To this end, the present invention relates to a virus formulation which contains at least one inhibitor of free radical oxidation which effectively enhances stability of the virus-containing formulation. While the exemplified adenovirus-based formulations such as A113 represent a preferred formulation, these formulations in no way suggest a limitation to additional formulations and methods of use based on alternative formulations components.

A third core embodiment of the present invention comprises inclusion, alone or in combination with free radical oxidation inhibitors, an effective amount of plasmid DNA, which is shown to effectively increase the long term stability of a virus formulation and conditions as described throughout this specification. Therefore, the present invention also relates to a virus formulation which contains an amount of a nucleic acid such that addition of the nucleic acid effectively enhances stability of the virus-containing formulation.

Another embodiment of the present invention are formulations which comprise components disclosed herein which result in liquid formulations which provide enhanced stability of adenoviruses representing multiple subgroups (e.g., such as B, C and/or D; alone or in combination, depending on the particular formulation); such formulations will be compatible with parenteral administration and provide for enhanced stability in liquid form for sustained periods of time during storage at 2-8.degree. C. and, thus, amenable to commercial application. These formulations include, but are not limited to formulations comprising relevant excipients in ranges as disclosed herein while being buffered to a lower pH (i.e., from about pH 6.0 to about pH 7.5), preferably by histidine, and being low in salt or even salt free, with the balance of osmolarity being contributed by one or more cryoprotectants, such as sucrose. To this end, the present invention also relates to methods of providing for liquid formulations for alternative (i.e., non-subgroup C adenovirus) and/or multiple adenovirus serotypes, whereby such formulations provide for increased virus stability over prolonged storage times. Such method include, but are not limited to, utilizing formulations disclosed in Example sections 15-18 (see Original Patent), as well as excipient combinations and/or excipient concentration ranges thereof.

The enhanced long-term stability up through the 2-8.degree. C. range results in an extended shelf life of the virus formulations disclosed herein, allowing for storage and eventual host administration of these liquid formulations over about a 1-2 year period with acceptable losses in virus infectivity. In addition, formulations of the present invention show stability through extended freeze/thaw cycles.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to formulations which stabilize a respective virus component and to related pharmaceutical products, preferably for use in gene therapy and/or vaccine applications. A preferred stabilized virus containing formulation disclosed herein is liquid adenovirus formulation, which shows improved stability when stored in about the 2-8.degree. C. range and higher while also being compatible with parenteral administration. These preferred formulations which are able to stabilize a respective virus (such as a recombinant adenovirus) may comprise a buffer, a sugar, a salt, a divalent cation, a non-ionic detergent, as well as additional components which enhance stability to the added virus, including but not limited to a free radical scavenger and/or a chelating agent (i.e., an inhibitor of free radical oxidation). In addition to excellent viral stability for prolonged periods of time at -70.degree. C. and -20.degree. C., the formulations which comprise various concentrations of adenovirus are amenable to prolonged storage at 2.degree. C. to 8.degree. C. and higher for periods up to at least one to two years. The virus formulations which may show enhanced long term storage stability include but are not necessarily limited to adenovirus, adeno-associated virus, retroviruses, herpes virus, vaccinia virus, rotovirus, pox viruses. The preferred virus is a human adenovirus, especially a serotype from a subgroup which shows negligible or no tumor growth in animals, such as subgroup C (Ad1, Ad2, Ad5 and Ad6), subgroup D (Ad8, Ad9, Ad10, Ad13, Ad15, Ad17, Ad19, Ad20, Ad22, Ad23, Ad24, Ad25, Ad26, Ad27, Ad28, Ad29, Ad30, Ad32, Ad33, Ad36, Ad37, Ad38, Ad39, Ad42, Ad43, Ad44, Ad45, Ad46, and Ad4), subgroup B (Ad3, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, and Ad35) and subgroup E (Ad4). For an exhaustive adenovirus classification scheme, see Fundamental Virology, 3.sup.rd Edition, Ch. 30 @ page 980, Ed. Fields, et al. 1996, Lippincott-Raven. Especially preferred serotypes are selected C serotypes Ad5, Ad2, and Ad6, subgroup D serotype Ad24, Ad26, Ad34 and Ad35. With the guidance provided by this specification, the skilled artisan may adapt the formulations disclosed herein to non-exemplified adenovirus serotypes as well as other viruses. To this end, the present invention relates to the use of these formulations to stabilize alternative purified virus, and to the compositions thereof.

The formulations of the present invention provide stability to adenovirus at varying degrees of virus concentration and may be administered to a variety of vertebrate organisms, preferably mammals and especially humans. The stabilized viral formulations of the present invention are preferably recombinant adenovirus-based compositions, wherein administer as a vaccine, for example, may offer a prophylactic advantage to previously uninfected individuals and/or provide a therapeutic effect by reducing viral load levels within an infected individual, thus prolonging the asymptomatic phase of a particular microbial infection, such as an HIV infection. A preferred aspect of the invention is a recombinant adenovirus formulation (i.e., an adenovirus containing a whole or a portion of a transgene which is expressed within the target host subsequent to host administration, such as in any mammalian/human gene therapy- or gene vaccination-based methodology available to the skilled artisan) which shows enhanced stability characteristics described herein with a virus concentration in the range from about 1.times.10.sup.7 vp/mL (virus particles/milliliter) to about 1.times.10.sup.13 vp/mL. A more preferred range is from about 1.times.10.sup.9 to 1.times.10.sup.12 vp/mL, with an especially preferred virus concentration being from about 1.times.10.sup.11 to 1.times.10.sup.12 vp/mL. Therapeutic, prophylactic or diagnostic compositions of the formulations of the present invention are administered to an individual in amounts sufficient to treat, prevent or diagnose the respective disorder. The effective amount for human administration may, of course, vary according to a variety of factors such as the individual's condition, weight, sex and age. Other factors include the mode of administration. The amount of expressible DNA to be administered to a human recipient will depend on the strength of the transcriptional and translational promoters used in the recombinant viral construct, and, if used as a vaccine, on the immunogenicity of the expressed gene product, as well as the level of pre-existing immunity to a virus such as adenovirus. The formulations of the present invention are optimally a buffered solution. It will be known to one of skill in the art to provide virus formulations of the present invention in a physiologically acceptable buffer, preferably but not necessarily limited to a formulation buffered with Tris (tromethamine), histidine, phosphate, citrate, succinate, acetate, glycine, and borate, within a pH range including but not limited to about 7.0 to about 9.0, preferably a pH range from about 7.5 to about 8.5. Tris is a preferred buffer in formulations disclosed herein (e.g., see Example sections 1-14) for this pH range; with histidine being a preferred buffering agent to provide for a formulation pH in a range from about 6.0 to about 7.5 (e.g., see Example sections 15-18).

An additional aspect of the formulations of the present invention relates to a formulation which comprises a minimal amount of at least one non-ionic surfactant added to reduce adsorption to container surfaces as well as possibly providing increased virus stabilization. Non-ionic surfactants for use in the formulations of the present invention include but are not limited to polyoxyethylene sorbitan fatty acid esters, including but not limited to Polysorbate-80 (Tween 80.RTM.), Polysorbate-60 (Tween 60.RTM.), Polysorbate-40 (Tween 40.RTM.) and Polysorbate-20 (Tween 20.RTM.), polyoxyethylene alkyl ethers, including but not limited to Brij 58.RTM., Brij 35.RTM., as well as others such as Triton X-100.RTM., Triton X-114.RTM., NP40.RTM., Span 85 and the Pluronic series of non-ionic surfactants (e.g., Pluronic 121).

An additional component which further stabilizes the added viral component comprise the addition of at least one salt of a divalent cation, including but not necessarily limited to MgCl.sub.2, CaCl.sub.2 and MnCl.sub.2. The preferred divalent cations are MgCl.sub.2 and CaCl.sub.2 at a concentration ranging from about 0.1 mM to about 5 mM.

Another component which contributes to virus stabilization over large temperature ranges and for prolonged storage periods is a cryoprotectant, especially at concentrations amenable to human administration. Cyroprotectants include but are not necessarily limited to addition of polyhydroxy hydrocarbons such as sorbitol (A443), mannitol (A442), glycerol and dulcitol and/or disaccharides such as sucrose, lactose, maltose or trehalose.

An additional component of the formulations of the present invention which enhance viral stability comprise a salt, including but not necessarily limited to sodium chloride, sodium sulfate, and ammonium sulfate, present at an ionic strength which is physiologically acceptable to the host. A purpose of inclusion of a salt in the formulation is to attain the desired ionic strength or osmolarity. Contributions to ionic strength may come from ions produced by the buffering compound as well as from the ions of non-buffering salts.

A centerpiece of the formulations of the present invention which enhance viral stability relate to inclusion of components that act as inhibitors of free radical oxidation. As noted throughout the specification, virus stability in a pharmaceutical formulation may be effected by the type of buffer, salt concentration, pH, light exposure, temperature storage and the such. It is also shown herein that components which may inhibit free radical oxidation further enhance the stability characteristics of the core adenoviral formulations disclosed herein. Free radical oxidation inhibitors which may be utilized include but are not necessarily limited to ethanol (EtOH), EDTA, an EDTA/ethanol combination, triethanolamine (TEOA), mannitol, histidine, glycerol, sodium citrate, inositol hexaphosphate, tripolyphosphate, succinic and malic acid, desferal, ethylenediamine-Di(o-hydoxy-phenylacetic acid (EDDHA) and diethylenetriaminepenta-acetic acid (DTPA), or specific combinations thereof. It is preferred that the inhibitor of free radical oxidation be either an EDTA/EtOH combination, EtOH alone, or triethanolamine (TEOA). It is shown herein that the combination with other components may determine the effectiveness of the free radical oxidation inhibitor. For example, the combination of EDTA/EtOH is shown to be very effective at increasing stability, while DTPA (alone) in the absence of MgCl.sub.2 also enhances stability. Therefore, the skilled artisan may "mix and match" various components, in some cases a scavenger and a chelator are required, while other formulations only a chelator may be required. Preferably, the choice of chelator will determine whether or not the addition of a scavenger is needed. Additional free radical scavengers and chelators are known in the art and apply to the formulations and methods of use described herein. It is disclosed herein that addition of such inhibitors of free radical oxidation results in a substantial increase in long term stability of liquid virus formulations. It is noted that the present invention is not limited to use of these excipients only in the preferred formulations described herein, but are in fact meant to include additional, non-exemplified virus formulations which will be amenable to increased stability within useful temperature ranges by the addition of one or more of these compounds.

The formulations of the present invention which enhance viral stability rely on a useful range of total osmolarity which both promotes long term stability at temperature of 2-8.degree. C., or higher, while also making the formulation useful for parenteral, and especially intramuscular, injection. To this end the effective range of total osmolarity (the total number of molecules in solution) is from about 200 mOs/L to about 800 mOs/L, with a preferred range from about 250 mOs/L to about 450 mOs/L. An especially preferred osmolarity for the formulations disclosed herein is about 300 mOs/L. Therefore, it will be apparent that the amount of a cyroprotectant, such as sucrose or sorbitol, will depend upon the amount of salt in the formulation in order for the total osmolarity of the solution to remain within an appropriate range. Therefore a salt free formulation may contain from about 5% to about 25% sucrose, with a preferred range of sucrose from about 7% to about 15%, with an especially preferred sucrose concentration in a salt free formulation being from 10% to 12%. Alternatively, a salt free sorbitol-based formulation may contain sorbitol within a range from about 3% to about 12%, with a preferred range from about 4% to 7%, and an especially preferred range is from about 5% to about 6% sorbitol in a salt-free formulation. Salt-free formulations will of course warrant increased ranges of the respective cryoprotectant in order to maintain effective osmolarity levels. To again utilize sucrose and sorbitol as examples, and not as a limitation, an effective range of a sucrose-based solution in 75 mM NaCl is from about 2% about 7.5% sucrose, while a sorbitol-based solution in 75 mM NaCl is from about 1% to about 4% sorbitol.

In view of the discussion above, the present invention relates to a formulation containing an adenovirus, such as a recombinant adenovirus for use in gene therapy and/or gene vaccination applications, with show increased viral stability properties and which at least contain a buffer, a salt, a sugar and a surfactant.

A particular embodiment of the present invention relates to such a recombinant adenovirus formulation which comprises Tris as the buffer, sucrose as the cryoprotectant, NaCl as the salt, MgCl.sub.2 as the divalent cation and either Polysorbate-80 or Polysorbate-40 as the surfactant.

In a particular embodiment of the present invention the formulation is buffered with Tris to a range from about pH 7.5 to about pH 8.5; sucrose is added within a range upwards of a weight to volume percentage of 10, depending upon the salt concentration; the salt being NaCl which is added at concentration within a range of upwards of 250 mM NaCl, complementing the sucrose concentration such that total osmolarity ranges from about 200 mOs/L to about 800 mOs/L; the divalent cation is MgCl.sub.2 in a range from about 0.1 mM to about 10 mM, and the surfactant is either Polysorbate-80 at a concentration from about 0.001% to about 1% or Polysorbate-40 at a concentration from about 0.001% to about 1%.

In a further embodiment of the present invention the formulation is buffered with about 1 mM to about 10 mM Tris to a range from about pH 7.5 to about pH 8.5; sucrose is present in a weight to volume range of about 2% to about 8% and NaCl is present from a range of about 25 mM to about 250 mM, the sucrose and NaCl concentrations being complementary such that the total osmolarity ranges from about 200 mOs/L to about 800 mOs/L; the divalent cation is MgCl.sub.2 in a range from about 0.1 mM to about 5 mM, and the surfactant is either Polysorbate-80 at a concentration from about 0.001% to about 0.25% or Polysorbate-40 at a concentration from about 0.001% to 0.25%.

In another embodiment of the present invention the formulation is buffered with about 2.5 mM to about 7.5 mM Tris to a pH of about 8.0; sucrose is present in a weight to volume range of about 2% to about 8% and NaCl is present from a range of about 25 mM to about 250 mM, the sucrose and NaCl contributing to a total osmolarity range from about 250 mOs/L to about 450 mOs/L; the divalent cation is MgCl.sub.2 in a range from about 0.5 mM to about 2.5 mM, and the surfactant is either Polysorbate-80 at a concentration from about 0.001% to about 0.1% or Polysorbate-40 at a concentration from about 0.001% to 0.1%.

In a further embodiment of the present invention the formulation is buffered with about 5.0 mM Tris to a pH of about 8.0; sucrose is present in a weight to volume range of about 4% to about 6% and NaCl is present from a range of about 50 mM to about 100 mM, the sucrose and NaC contributing to a total osmolarity range from about 250 mOs/L to about 450 mOs/L; the divalent cation is MgCl.sub.2 in a range from about 1 mM to about 2 mM, and the surfactant is either Polysorbate-80 at a concentration from about 0.001% to about 0.1% or Polysorbate-40 at a concentration from about 0.001% to 0.1%.

In a still further embodiment of the present invention the formulation is buffered with about 5.0 mM Tris, at pH 8.0; sucrose is present in a weight to volume of about 5%; NaCl is present at about 75 mM, with the total osmolarity at about 300 mOs/L; MgCl.sub.2 in at about 1 mM to 2 mM, and either Polysorbate-80 is present at a concentration of about 0.02% or Polysorbate-40 at a concentration of about 0.005%.

An exemplified portion of the present invention the formulation is buffered with about 5.0 mM Tris, at pH 8.0; sucrose is present in a weight to volume of 5% (146 mM); NaCl is present at 75 mM, with the total osmolarity approximately 310 mOs/L; MgCl.sub.2 at 1 mM, and Polysorbate-80 is present at a concentration of 0.005%. This formulation is herein designated A105.

Another exemplification shows an effective PS-80 range to at least 0.1%, as opposed to A105, where PS-80 is found at 0.005%. This formulation is buffered with about 5.0 mM Tris, at pH 8.0; sucrose is present in a weight to volume of 5% (146 MM); NaCl is present at 75 mM, MgCl.sub.2 is at 1 mM, and Polysorbate-80 is present at a concentration of 0.1%. This formulation is herein designated A111.

Another embodiment of the present invention is exemplified by a formulation buffered with about 5.0 mM Tris, at pH 8.0; sucrose is present in a weight to volume of 5% (146 mM); NaCl is present at 75 mM, MgCl.sub.2 at 1 mM, and Polysorbate-40 is present at a concentration of 0.005%. This formulation is herein designated A128, as shown in Example 1 (see Original Patent).

Yet another exemplification is a formulation identical to A128, except that Polysorbate-40 is present at a concentration of 0.1%, showing an effective range of Polysorbate-40. This formulation is herein designated A129, as shown in Example 1.

The present invention further relates recombinant adenovirus formulations which omit at least one component of the above-disclosed component, including but not limited to formulation A108 (no divalent cation) or formulation A109 (no surfactant).

An essential quality of the present invention is the finding that non-reducing free radical scavengers and/or chelators are important for maximizing both short and long term stability of viral formulations, especially recombinant adenoviral formulations disclosed herein. To this end, and as noted above, a critical preferred embodiment of the present invention is a viral formulation which contains one or more components which act as an inhibitor of free radical oxidation. It is shown herein that addition of these components enhance long term stability at temperatures up through the 2-8.degree. C. range, or higher, when compared to core formulations which do not contains these inhibitors. In addition, these formulations are compatible with parenteral administration. The increased stability of these formulations shows that oxidation is a major pathway of adenovirus inactivation which results in a loss of infectivity during storage.

The present invention relates to a recombinant adenoviral formulation buffered with Tris to a range from about pH 7.5 to about pH 8.5; sucrose is added within a range upwards of a weight to volume percentage of 10, depending upon the salt concentration; the salt being NaCl which is added at concentration within a range of upwards of 250 mM NaCl, complementing the sucrose concentration such that total osmolarity ranges from about 200 mOs/L to about 800 mOs/L; the divalent cation is MgCl.sub.2 in a range from about 0.1 mM to about 10 mM, and the surfactant is either Polysorbate-80 at a concentration from about 0.001% to about 2% or Polysorbate-40 at a concentration from about 0.001% to about 1%, wherein the formulation further comprises one or more components described herein which inhibit free radical oxidation, including but not limited to ethanol (EtOH), EDTA, an EDTA/ethanol combination, triethanolamine (TEOA), mannitol, histidine, glycerol, sodium citrate, inositol hexaphosphate, tripolyphosphate, succinic and malic acid, desferal, ethylenediamine-Di(o-hydoxy-phenylacetic acid (EDDHA) and diethylenetriaminepenta-acetic acid (DTPA).

In a further embodiment of the present invention the formulation is buffered with about 1 mM to about 10 mM Tris to a range from about pH 7.5 to about pH 8.5; sucrose is present in a weight to volume range of about 2% to about 8% and NaCl is present from a range of about 25 mM to about 250 mM, the sucrose and NaCl concentrations being complementary such that the total osmolarity ranges from about 200 mOs/L to about 800 mOs/L; the divalent cation is MgCl.sub.2 in a range from about 0.1 mM to about 5 mM, and the surfactant is either Polysorbate-80 at a concentration from about 0.001% to about 0.25% or Polysorbate-40 at a concentration from about 0.001% to 0.5%, wherein the formulation further comprises one or more components described herein which inhibit free radical oxidation, including but not limited to ethanol (EtOH), EDTA, an EDTA/ethanol combination, triethanolamine (TEOA), mannitol, histidine, glycerol, sodium citrate, inositol hexaphosphate, tripolyphosphate, succinic and malic acid, desferal, ethylenediamine-Di(o-hydoxy-phenylacetic acid (EDDHA) and diethylenetriaminepenta-acetic acid (DTPA).

In a specific embodiment of the present invention the formulation is buffered with about 2.5 mM to about 7.5 mM Tris to a pH of about 8.0; sucrose is present in a weight to volume range of about 2% to about 8% and NaCl is present from a range of about 25 mM to about 250 mM, the sucrose and NaCl contributing to a total osmolarity range from about 250 mOs/L to about 450 mOs/L; the divalent cation is MgCl.sub.2 in a range from about 0.5 mM to about 2.5 mM, and the surfactant is either Polysorbate-80 at a concentration from about 0.001% to about 0.1% or Polysorbate-40 at a concentration from about 0.001% to 0.05%, wherein the formulation further comprises one or more components described herein which inhibit free radical oxidation, including but not limited to ethanol (EtOH), EDTA, an EDTA/ethanol combination, triethanolamine (TEOA), mannitol, histidine, glycerol, sodium citrate, inositol hexaphosphate, tripolyphosphate, succinic and malic acid, desferal, ethylenediamine-Di(o-hydoxy-phenylacetic acid (EDDHA) and diethylenetriaminepenta-acetic acid (DTPA).

In another embodiment of the present invention the formulation is buffered with about 5.0 mM Tris to a pH of about 8.0; sucrose is present in a weight to volume range of about 4% to about 6% and NaCl is present from a range of about 50 mM to about 100 mM, the sucrose and NaCl contributing to a total osmolarity range from about 250 mOs/L to about 450 mOs/L; the divalent cation is MgCl.sub.2 in a range from about 1 mM to about 2 mM, and the surfactant is either Polysorbate-80 at a concentration from about 0.001% to about 0.1% or Polysorbate-40 at a concentration from about 0.001% to 0.01%, wherein the formulation further comprises one or more components described herein which inhibit free radical oxidation, including but not limited to ethanol (EtOH), EDTA, an EDTA/ethanol combination, triethanolamine (TEOA), mannitol, histidine, glycerol, sodium citrate, inositol hexaphosphate, tripolyphosphate, succinic and malic acid, desferal, ethylenediamine-Di(o-hydoxy-phenylacetic acid (EDDHA) and diethylenetriaminepenta-acetic acid (DTPA).

In a still further embodiment of the present invention the formulation is buffered with about 5.0 mM Tris, at pH 8.0; sucrose is present in a weight to volume of about 5%; NaCl is present at about 75 mM, with the total osmolarity at about 300 mOs/L; MgCl.sub.2 in at about 1 mM, and either Polysorbate-80 is present at a concentration of about 0.02% or Polysorbate-40 at a concentration of about 0.005%, wherein the formulation further comprises one or more components described herein which inhibit free radical oxidation, including but not limited to ethanol (EtOH), EDTA, an EDTA/ethanol combination, triethanolamine (TEOA), mannitol, histidine, glycerol, sodium citrate, inositol hexaphosphate, tripolyphosphate, succinic and malic acid, desferal, ethylenediamine-Di(o-hydoxy-phenylacetic acid (EDDHA) and diethylenetriaminepenta-acetic acid (DTPA).

In the above-described formulations, at least one non-reducing free radical scavenger may be added to concentrations which effectively enhance stability of the core formulation. Especially useful ranges include (i) EDTA from about 1 .mu.M to about 500 .mu.M; with some formulations containing EDTA at concentrations up to 1000 .mu.M (A461 at 750 .mu.M), preferably in a range from about 50 .mu.M to about 250 .mu.M, and an especially preferred concentration of at or around 100 .mu.M; (ii) ethanol from about 0.1% to about 5.0%, preferably in a range from about 0.25% to about 2.0%, and an especially preferred amount totaling at or around 0.5%; (iii) DTPA from about 1 .mu.M to about 500 .mu.M, preferably in a range from about 50 .mu.M to about 250 .mu.M, and an especially preferred concentration at or around 100 .mu.M; (iv) CaCl.sub.2 from about 0.1 mM to about 10 mM, preferably in a range from about 0.5 mM to about 5 mM, and an especially preferred concentration at or around 1 mM; and, (v) sodium citrate from about 1 mM to about 100 mM, preferably in a range from about 5 mM to about 25 mM, and an especially preferred concentration at or around 10 mM. These inhibitors of free radical oxidation may also be added in various combinations, including but not limited to two scavengers (e.g., 113), a sole (e.g., A114), or possible a sole scavenger in the absence of another component, such as a divalent cation (e.g., A116).

In another embodiment of the formulation is buffered with about 5.0 mM Tris, at pH 8.0; sucrose is present in a weight to volume of 5% (146 mM); NaCl is present at 75 mM, with the total osmolarity approximately 400 mOs/L; MgCl.sub.2 at 1 mM, and Polysorbate-80 is present at a concentration of 0.005%, EDTA is present at 100 .mu.M and ethanol at 0.5%. This formulation is designated A113, as shown in Example 1.

In an additional embodiment the formulation is buffered with about 5.0 mM Tris, at pH 8.0; sucrose is present in a weight to volume of 5% (146 mM); NaCl is present at 75 mM, with the total osmolarity approximately 310 mOs/L; MgCl.sub.2 at 1 mM, and Polysorbate-80 is present at a concentration of 0.005%, and triethanolamine (TEOA) is present at 1 mM. This formulation is herein designated A114, as shown in Example 1.

In another embodiment the formulation is buffered with about 5.0 mM Tris, at pH 8.0; sucrose is present in a weight to volume of 5% (146 mM); NaCl is present at 75 mM, with the total osmolarity approximately 350 mOs/L; MgCl.sub.2 at 1 mM, and Polysorbate-80 is present at a concentration of 0.005%, and sodium citrate at 10 mM. This formulation is herein designated A115, also as shown in Example 1.

In still another embodiment the formulation is buffered with about 5.0 mM Tris, at pH 8.0; sucrose is present in a weight to volume of 5% (146 mM); NaCl is present at 75 mM, Polysorbate-80 is present at a concentration of 0.005%, and DTPA at 100 .mu.M. This formulation is herein designated A116, also as shown in Example 1.

In another embodiment of the present invention the formulation is buffered with about 5.0 mM Tris-HCl, at pH 8.0; sucrose is present in a weight to volume range of 5% (146 mM); NaCl is present at 75 mM, MgCl.sub.2 at 1 mM, and Polysorbate-80 is present at a concentration of 0.005%, and mannitol is present at 3% (w/v). This formulation is herein designated A121.

In yet another embodiment the formulation is buffered with about 5.0 mM Tris, at pH 8.0; sucrose is present in a weight to volume of 5% (146 mM); NaCl is present at 75 mM, MgCl.sub.2 at 1 mM, Polysorbate-80 is present at a concentration of 0.005%, and ethanol is present at a concentration of 0.5% (A132) and 1.0% (A134). These two formulations are disclosed in Example 1.

Another embodiment shows a formulation buffered with about 5.0 mM Tris, at pH 8.0; sucrose is present in a weight to volume of 5% (146 mM); NaCl is present at 75 mM, with the total osmolarity approximately 310 mOs/L; MgCl.sub.2 at 1 mM, Polysorbate-80 is present at a concentration of 0.005%, and EDTA at 100 .mu.M. This formulation is herein designated A133, also as shown in Example 1.

Another preferred embodiment shows a formulation buffered with about 5.0 mM Tris, at pH 8.0; sucrose is present in a weight to volume of 5% (146 mM); NaCl is present at 75 mM, with the total osmolarity approximately 500 mOs/L; MgCl.sub.2 at 1 mM, Polysorbate-80 is present at a concentration of 0.005%, EDTA at 100 .mu.M and ethanol 1.0%. This formulation is herein designated A135, also as shown in Example 1.

In another embodiment of the present invention the formulation is buffered with about 5.0 mM Tris, at pH 8.0; sucrose is present in a weight to volume of 5% (146 mM); NaCl is present at 75 mM, with the total osmolarity approximately 400 mOs/L; MgCl.sub.2 at 1 mM, Polysorbate-80 is present at a concentration of 0.1%, EDTA at 100 .mu.M and ethanol 0.5%. This formulation is herein designated A136, also shown in Example 1.

As noted above, it is also within the scope of the present invention to substitute a preferred divalent cation such as MgCl.sub.2 with a difference divalent cation, CaCl.sub.2. Such a substitution may relate to any of the formulations disclosed herein. An example of such a substitution is formulation A120, which comprises 5.0 mM Tris-HCl, at pH 8.0; sucrose is present in a weight to volume of 5% (146 mM); NaCl is present at 75 mM, Polysorbate-80 at 0.005%, EDTA at 100 .mu.M, ethanol at 0.5% and CaCl.sub.2 at 1 mM. Formulation A120 is shown in Example 1.

The present invention further relates recombinant adenovirus formulations which omit at least one component of the above-disclosed components, including but not limited to formulation A116 (excipient: DTPA at 100 .mu.M; no divalent cation), formulation A117 (excipients: EDTA at 100 .mu.M and EtOH at 0.5%; no divalent cation), formulation A459 and A461 (excipient: EDTA at 250 .mu.M and 750 .mu.M, respectively, no divalent cation) formulation A118 (triethanolamine at 1.0 mM; no divalent cation), formulation A119 (excipient: sodium citrate at 10 mM; no divalent cation).

In addition to the above-disclosed excipients which act as inhibitor of free radical oxidation, the present invention further relates to a recombinant viral formulation which additionally comprises plasmid DNA at a concentration from about 0.01 mg/ml to about 10 mg/ml. The addition of plasmid DNA effectively increases the stability of a recombinant virus formulation, such as the recombinant adenovirus exemplified herein. Therefore, plasmid DNA may be added to any core formulations (e.g., A105 and A128, which do not contain additional excipients such as free radical oxidation inhibitors), as well as core formulations comprising such excipients. A preferred concentration range for the plasmid DNA is from about 0.5 mg/l to about 5.0 mg/ml, with an additionally preferred plasmid DNA concentration at 1 mg/ml.

In another embodiment of the present invention the formulation is buffered with about 5.0 mM Tris-HCl, at pH 8.0; sucrose is present in a weight to volume range of 5% (146 mM); NaCl is present at 75 mM, MgCl.sub.2 at 1 mM, and Polysorbate-80 is present at a concentration of 0.005%, and plasmid DNA is present at 1 mg/ml. This formulation is herein designated A137.

In still another embodiment of the present invention the formulation is buffered with about 5.0 mM Tris, at pH 8.0; sucrose is present in a weight to volume of 5% (146 mM); NaCl is present at 75 mM, with the total osmolarity approximately 400 mOs/L; MgCl.sub.2 at 1 mM, and Polysorbate-80 is present at a concentration of 0.005%, EDTA is present at 100 .mu.M, ethanol is present at 0.5% and plasmid DNA is present at 1 mg/mL. This formulation is designated A138, as shown in Example 1.

In another preferred embodiment of the present invention the formulation is buffered with about 5.0 mM Tris, at pH 8.0; mannitol is present in a weight to volume of 2.7% (147 mM); NaCl is present at 75 mM, with the total osmolarity approximately 400 mOs/L; MgCl.sub.2 at 1 mM, and Polysorbate-80 is present at a concentration of 0.005%, EDTA is present at 100 .mu.M and ethanol at 0.5%. This formulation is designated A149, as shown in Example 1.

Another embodiment shows a formulation buffered with about 5.0 mM Tris, at pH 8.0; sucrose is present in a weight to volume of 5% (146 mM); NaCl is present at 75 mM, with the total osmolarity approximately 400 mOs/L; MgCl.sub.2 at 1 mM, and Polysorbate-80 is present at a concentration of 0.005%, EDTA is present at 100 .mu.M, ethanol is present at 0.5% and histidine is present at 5 mM. This formulation is designated A151a, as shown in Example 1.

Another embodiment of the present invention disclosed a formulation buffered with about 5.0 mM Tris, at pH 7.5 at 30.degree. C.; while sucrose is present in a weight to volume of 5% (146 mM); NaCl is present at 75 mM, with the total osmolarity approximately 400 mOs/L; MgCl.sub.2 at 1 mM, and Polysorbate-80 is present at a concentration of 0.005%, EDTA is present at 100 .mu.M, ethanol is present at 0.5% and histidine is present at 5 mM. This formulation is designated A151b, as shown in Example 1.

In another embodiment of the present invention the formulation is buffered with about 5.0 mM Tris, at pH 7.5 at 30.degree. C.; sucrose is present in a weight to volume of 5% (146 mM); NaCl is present at 75 mM, with the total osmolarity approximately 400 mOs/L; MgCl.sub.2 at 2 mM, and Polysorbate-80 is present at a concentration of 0.005%, EDTA is present at 100 .mu.M, ethanol is present at 0.5%, histidine is present at 5 mM and triethanolamine is present at 5 mM. This formulation is designated A152, as shown in Example 1.

In still another embodiment of the present invention the formulation is buffered with about 5.0 mM Tris, at pH 7.5 at 30.degree. C.; sucrose is present in a weight to volume of 5% (146 mM); NaCl is present at 75 mM, with the total osmolarity approximately 400 mOs/L; MgCl.sub.2 at 2 mM, and Polysorbate-80 is present at a concentration of 0.005%, EDTA is present at 100 .mu.M, ethanol is present at 0.5%, histidine is present at 5 mM, triethanolamine is present at 5 mM and glycerol is present at 5% (v/v). This formulation is designated A153, as shown in Example 1. As noted above, the dosage regimen utilizing the compounds of the present invention is selected in accordance with a variety of factors including type, level of pre-existing immunity to adenovirus, species, age, weight, sex and medical condition of the patient; the severity of the condition to be treated; the route of administration; the renal, hepatic and cardiovascular function of the patient; and the particular compound thereof employed. A physician or veterinarian of ordinary skill can readily determine and prescribe the effective amount of the drug required to prevent, counter or arrest the progress of the condition. Optimal precision in achieving concentrations of drug within the range that yields efficacy without toxicity requires a regimen based on the kinetics of the drug's availability to target sites. This involves a consideration of the distribution, equilibrium, and elimination of a drug.

The formulated recombinant viruses described herein may also be formulated with an adjuvant or adjuvants which may increase immunogenicity of the expressed transgene. A number of these adjuvants are known in the art and are available for use, including but not limited to saponin, monophosphoryl lipid A, non-ionic block copolymers composed of polyoxyethylene and polyoxypropylene or other compounds which increase immunogenicity of expressed transgene. Another adjuvant for use with the recombinant viruses described herein are one or more forms of an aluminum phosphate-based adjuvant wherein the aluminum phosphate-based adjuvant possesses a molar PO.sub.4/Al ratio of approximately 0.9. An additional mineral-based adjuvant may be generated from one or more forms of a calcium phosphate. These mineral-based compounds for use as DNA vaccines adjuvants are disclosed in PCT International Application No. PCT/US98/02414 (WO 98/35562), which is hereby incorporated by reference.

The recombinant virus formulations described herein are administered to the vertebrate host (preferably a mammalian host and especially a human recipient) by any means known in the art, such as enteral and parenteral routes. These routes of delivery include but are not limited to intramusclar injection, intraperitoneal injection, intravenous injection, inhalation or intranasal delivery, oral delivery, sublingual administration, subcutaneous administration, transdermal administration, transcutaneous administration, percutaneous administration or any form of particle bombardment, such as a biolostic device such as a "gene gun" or by any available needle-free injection device. The preferred methods of delivery of the recombinant viruses described herein are intramuscular injection and needle-free injection. An especially preferred method is intramuscular delivery.

Another embodiment of the present invention relates to formulations comprising components disclosed herein which provide for enhanced adenovirus stability for multiple subgroups (e.g., such as B, C and/or D; alone or in combination, depending on the particular formulation). These formulations are compatible with parenteral administration, stable in liquid form for sustained periods of time during storage at 2-8.degree. C. and, thus, amenable to commercial application. Such formulations include, but are not limited to formulations comprising excipients in ranges as disclosed herein while being buffered to a lower pH range by, for example, histidine, and being low in salt or even salt free, with the balance of osmolarity being contributed by a cryoprotectant, such as sucrose. More specifically, this portion of the invention embodies liquid virus formulations (including but not limited to recombinant adenovirus-based formulations) which are preferably buffered by histidine in a range from about pH 6.0 to pH 7.5; containing NaCl in a range from no salt up to about 75 mM-100 mM, with a preference to up to about 50 mM (e.g., as exemplified in several formulations as a NaCl concentration at 35 mM); a cryoprotectant (including but not limited to sucrose) from about 7.5% to 25%; with MgCl.sub.2, polysorbate (including but not limited to PS-80), EDTA and/or ethanol within ranges disclosed herein. Such formulations include but are not limited to histidine-buffered virus formulations listed in Example 15 (see Original Patent), and any such excipient combination and/or excipient concentration range thereof.

It is shown herein that manipulation of sugar/salt (e.g., sucrose/NaCl) concentrations and/or reduction of pH may offer an enhanced level for stabilizing alternate (i.e., non subgroup C adenovirus) or multiple adenovirus serotypes. As shown in Example 17 (see Original Patent), formulations A423, A424, A428 and A429 show enhanced stability of serotype B adenovirus. Therefore, virus formulations which may be especially useful for providing enhanced stability of Ad B serotypes will be a formulation with a virus concentration in the range from about 1.times.10.sup.7 vp/mL to about 1.times.10.sup.13 vp/mL and a total osmolarity in a range from about 200 mOs/L to about 800 mOs/L. This formulation is preferably buffered by histidine at a pH from about 6.0 to about 7.5 and also contains sucrose in a weight to volume range from about 7.5% to about 25% (w/v); NaCl up to about 75 mM, MgCl.sub.2 at about 1 mM to 2 mM, either Polysorbate-80, Polysorbate-40 or Polysorbate-20 at a concentration from about 0.001%, with EDTA at about 100 .mu.M and ethanol at about 0.5% (w/v). It will be evident from review of this specification that narrower ranges may be applicable to preferred applications, including but not limited to the exclusion of NaCl from the formulation coupled with an increase in sucrose concentration, to a range from about 10% to 25% (w/v) sucrose, with a preference for the higher end of range for serotype B adenoviruses. To this end, a portion of the present invention relates to a virus formulation, especially an adenovirus B serotype virus formulation buffered to a pH range from about 6.0 to about pH 7.5 and comprising purified virus, at least one inhibitor of free radical oxidation selected from the group consisting of ethanol, EDTA, histidine, triethanolamine, and sodium citrate; and, at least one sugar (e.g., sucrose) at a combined weight to volume percentage from about 7.5% to about 25% w/v and optionally a salt (e.g., NaCl) up to a combined concentration of about 100 mM, such that the total osmolarity of the formulation is a range from about 200 mOs/L to about 800 mOs/L. These formulations may further comprise a non-ionic detergent selected, especially a non-ionic detergent selected from the group consisting of Polysorbate-80, Polysorbate-40 and Polysorbate-20 at a concentration range from about 0.001% to about 2%; and/or a divalent cation selected from the group consisting of MgCl.sub.2 and CaCl.sub.2 in an amount from about 0.1 mM to about 5 mM; and/or EDTA from about 1 .mu.M to about 1000 .mu.M and ethanol from about 0.1 to 1.0% (w/v). A423, A424, A428, and A429 exemplify this group of Ad-B stabilizing formulations. To this end, the present invention also relates to a method of preserving a subgroup B adenovirus which comprises generating a formulation comprising a combination and range of components discussed within this paragraph.

As shown in Example 18 (see Original Patent), formulations A442, A443, A444, A448, A459 and A461 show enhanced stability for serotype D adenovirus. Such a formulation is buffered at a pH range from about pH 6.0 to about pH 9.0; and further comprises sucrose in a weight to volume range from up to about 15% (w/v); NaCl up to about 75 mM, MgCl.sub.2 at about 1 mM to 2 mM, either Polysorbate-80, Polysorbate-40 or Polysorbate-20 at a concentration from about 0.001%, with EDTA at about 100 .mu.M, ethanol at about 0.5% (w/v) and histidine at about 10 mM. To this end, the present invention relates in part to a formulation comprising a purified virus, especially an adenovirus, having an adenovirus concentration in the range from about 1.times.10.sup.7 vp/mL to about 1.times.10.sup.13 vp/rnL and a total osmolarity in a range from about 200 mOs/L to about 800 mOs/L which is preferably buffered with Tris to a pH from about 6.0 to about 7.5, further comprising sucrose in a weight to volume range from greater than 5% to about 25% (w/v); NaCl up to about 50 mM, MgCl.sub.2 at about 1 mM to 2 mM, either Polysorbate-80, Polysorbate-40 or Polysorbate-20 at a concentration from about 0.001% to about 2%, EDTA from about 1 .mu.M to about 1000 .mu.M, ethanol from about 0.1 to 1.0% (w/v) and histidine from about 5mM to about 10 mM. Such serotype D-based formulations may optionally include mannitol at a concentration up to about 250 mM (A442), sorbitol at a concentration up to about 250 mM (A443) and/or triethanolamine at a concentration up to about 5 mM (A448). Additionally, the present invention also relates to a method of preserving a subgroup D adenovirus which comprises generating a formulation comprising a combination and range of components discussed within this paragraph.

It is also disclosed herein that certain formulations have the ability to provide enhanced adenovirus stability across multiple serotypes, such as A438, which provides enhanced adenovirus stability for serotype B, C and D virus. A A438-type formulation will preferably be a histidine buffered formulation containing a least one additional inhibitor of free radical oxidation selected from the group consisting of ethanol, EDTA, histidine, triethanolamine, and sodium citrate, with sucrose being present up to about a volume percentage up to about 10%, with a preference in the 5%-10% range, and sodium chloride up to about 50 mM, such that the total osmolarity of the formulation is a range from about 200 mOs/L to about 800 mOs/L. Additional excipients are as disclosed herein, namely MgCl.sub.2 at about 1 mM to 2 mM, Polysorbate-80, Polysorbate-40 or Polysorbate-20 at about 0.1%, EDTA at about 100 .mu.M, and ethanol at about 0.5% (w/v). Therefore, the present invention relates in part to a virus formulation wherein the purified virus is preferably adenovirus. As noted throughout this application, such a formulation has an adenovirus concentration in the range from about 1.times.10.sup.7 vp/mL to about 1.times.10.sup.13 vp/mL and a total osmolarity in a range from about 200 mOs/L to about 800 mOs/L. This type of adenovirus formulation is buffered to a pH range of about pH 6.0 to about pH 7.5 (preferably with histidine)and may further comprise a sugar, such as sucrose, in a weight to volume range from greater than 5% to about 10% (w/v); a salt, such as NaCl, up to about 50 mM, MgCl.sub.2 at about 1 mM to 2 mM, either Polysorbate-80, Polysorbate-40 or Polysorbate-20 at a concentration from about 0.001% to about 2%, EDTA from about 1 .mu.M to about 1000 .mu.M and ethanol from about 0.1 to 1.0% (w/v). An exemplified multiple serotype adenovirus formulation is disclosed herein as formulation A438. The present invention also relates to a method of preserving a multiple serotype adenovirus formulation (e.g., A438 for a subtype B, C and D-based adenovirus formulation) by generating a formulation comprising a combination and range of components discussed within this paragraph.

In accordance with the formulation compositions disclosed herein, the present invention also relates to methods of stabilizing virus formulation which comprises generating virus-containing formulations disclosed herein, such formulations which result in improved viral stability when stored in about the 2-8.degree. C. range and higher while also being compatible with parenteral administration, especially parenteral administration to humans. Therefore, these prescribed methods relate to the disclosed, and especially, the exemplified virus-containing formulations of the present invention. In addition, the present invention relates to a method of stabilizing a virus formulation which comprises adding at least one inhibitor of free radical oxidation to the formulation, such that the resultant formulation shows improved stability in about the 2-8.degree. C. range and higher while also being compatible with parenteral administration. Also, the present invention relates to a method of stabilizing a virus formulation which comprises adding a nucleic acid to the formulation, such that the resultant formulation also shows improved stability in about the 2-8.degree. C. range and higher while also being compatible with parenteral administration. Therefore, the present invention relates to a method of stabilizing a virus formulation which comprises preserving the virus of interest, preferably a recombinant virus, in any of the formulations described herein, and especially methods which comprise preservation of the virus by addition of at least one inhibitor of free radical oxidation and/or addition a nucleic acid to the formulation, such that the resultant formulation also shows improved stability in about the 2-8.degree. C. range and higher while also being compatible with parenteral administration. The present invention also relates to methods of providing for liquid formulations for alternative (i.e., non-subgroup C adenovirus) and/or multiple adenovirus serotypes, whereby such formulations provide for increased virus stability over prolonged storage times. Such methods prefer, but are not limited to, utilizing formulations disclosed in Example 15, as well as excipient combinations and/or excipient concentration ranges that the artisan will understand subsequent to a review of the formulations listed in Example 15.
 

Claim 1 of 26 Claims

1. A virus formulation, comprising: a) a purified adenovirus; b) at least one inhibitor of free radical oxidation selected from the group consisting of ethanol, EDTA, histidine, triethanolamine, and sodium citrate; and, c) at least one sugar at a combined weight to volume percentage from about 7.5% to about 25% w/v and optionally a salt up to a combined concentration of about 100 mM, such that the total osmolarity of the formulation is a range from about 200 mOs/L to about 800 mOs/L, wherein the formulation is buffered at a pH from about 6.0 to 7.0.
 

____________________________________________
If you want to learn more about this patent, please go directly to the U.S. Patent and Trademark Office Web site to access the full patent.