Cancer therapy with a parvovirus combined with an HDAC inhibitor

Abstract

Described is a pharmaceutical composition comprising (a) a parvovirus and (b) a histone deacetylase inhibitor (HDACI) and the use of said composition for treatment of cancer, e.g., brain tumor, cervical carcinoma, or pancreatic carcinoma.

Claims

1. A method of treating cervical carcinoma or pancreatic carcinoma comprising the administration of an effective dose of a pharmaceutical composition containing (a) a H-1 parvovirus (H-1PV) and (b) a histone deacetylase inhibitor (HDACI) selected from the group consisting of valproic acid (VPA) and suberoylanilide hydroxamic acid (SAHA), wherein said effective dose comprises an amount sufficient to lead to the reduction or remission of said cervical carcinoma or pancreatic carcinoma, wherein (a) the H-1 parvovirus (H-1PV) and (b) the HDACI are sequentially administered.

2. The method of claim 1, wherein the H-1 parvovirus (H-1PV) (a) is administered prior to the HDACI (b).

3. The method of claim 1, wherein said H-1 parvovirus (H-1PV) is administered by intratumoral administration.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: H-1PV and HDAC inhibitors kill cervical carcinoma derived cell lines in a synergistic manner

(2) HeLa (HPV 18 positive cell line) and SiHa (HPV 16 positive) were infected with H-1PV at the indicated MOIs and subsequently grown in the presence (black bar) or absence (grey bar) of HDACIs. The HDACIs tested were NaB (1 mM), TSA (50 nM) and SAHA (50 nM) for HeLa and NaB (2 mM), TSA (100 nM) and SAHA (500 nM) for SiHa cells. After incubation cell lysis was evaluated by measuring the release of lactate dehydrogenase (LDH). The values above bars (expressed in %) indicate synergistic effects obtained. Results from one representative experiment are shown.

(3) FIG. 2: H-1PV and HDAC inhibitors kill cervical carcinoma derived cells in a synergistic manner

(4) Primary cervical cancer cells were infected with H-1PV at the indicated MOIs and subsequently grown in the presence (black bar) or absence (grey bar) of HDACIs. The HDACIs tested were NaB (1 mM), TSA (100 nM) and SAHA (200 nM). After incubation, cell lysis was evaluated by measuring the release of lactate dehydrogenase (LDH). The values (%) indicate synergistic effects obtained. Results from one representative experiment are shown.

(5) FIG. 3: HDAC inhibitors enhance H-1 PV cytotoxicity

(6) (A) Either mock treated or H-1PV (MOI: 1 pfu/cell) infected HeLa cells were grown in the presence or absence of NaB (1 mM). After 48 h, cells were collected for the analysis of sub-G1 cell population by flow cytometry. The co-treatment with NaB increases the sub-GI cell population induced by H-1PV, indicating that the compound acts synergistically with the virus in killing cancer cells.

(7) (B) A summary of three independent experiments each performed in triplicate is presented. Bars represent mean values +/ relative standard deviations. P<0.05, paired Student's t tests are indicated (***).

(8) FIG. 4: H-1PV/HDAC inhibitors co-treatment increases the fraction of cells undergoing apoptosis

(9) The increase of the sub-G1 cell population, labeled as a blue line and indicated by numbers, indicates that HDAC inhibitors enhance H-1PV cytotoxic properties.

(10) FIG. 5: H-1PV and HDAC inhibitors kill cervical carcinoma derived cell lines in a synergistic manner.

(11) Cervical cancer derived cell lines (HeLa, CaSki, SiHa) and non-established cervical cancer cell cultures (CxCa) were infected with H-1PV at the indicated MOIs (pfu/cell) and grown for 72 h in the presence (grey bars) or absence (white bars) of VPA (1 mM). After incubation, cell lysis was evaluated by measuring the release of lactate dehydrogenase (LDH). Hatched bars at the right hand side of each chart show the minimal dose of H-1PV able to kill 90-100% of the cancer cells as monotherapy. Similar cytotoxicity was obtained at lower viral MOIs, by combining H-1PV with a sub-lethal dose of VPA (1 mM), indicating that both agents synergistically cooperate in killing cancer cells.

(12) FIG. 6: H-1PV/VPA co-treatment leads to complete regression of HeLa established tumors.

(13) 510.sup.6 HeLa cells were subcutaneously injected in the right flank of 5 weeks-old female nude rats. After 5 days (when tumour reached the volume of 200-400 mm.sup.3), tumour-bearing animals were randomized in four groups (n=8). Groups were treated either with DMEM (control), VPA (100 mk/kg), H-1PV (total dose of 1.25 109 pfu/animal, fractionated in 4 intratumoral administrations at days 5, 9, 16 and 23 post implantation), or a combination of both agents. Tumour volume was measured with a digital calliper on the days indicated and calculated according to the formula: volume (cm.sup.3)=LWH (length L, cm; width W, cm, height H, cm). Data shown represents the average values with standard deviation bars.

(14) FIG. 7: H-1PV and HDAC inhibitors kill PDAC derived cells in a synergistic manner.

(15) APC-1 and Capan-1 cells were plated in 96 wells plates (2000 cells/well). After 16 hours, cell were infected with H-1PV at the indicated MOI and grown in the presence or absence of VPA for additional 72 hours. Cellular lysis was measured by LDH assay.

(16) The present invention provides a pharmaceutical composition containing (a) a parvovirus and (b) a HDCA inhibitor, preferably as separate entities, e.g. in separate containers.

(17) Preferably, in said pharmaceutical composition the parvovirus and the HDCA inhibitor are present in an effective dose and combined with a pharmaceutically acceptable carrier. Pharmaceutically acceptable is meant to encompass any carrier, which does not interfere with the effectiveness of the biological activity of the active ingredients and that is not toxic to the patient to whom it is administered. Examples of suitable pharmaceutical carriers are well known in the art and include phosphate buffered saline solutions, water, emulsions, such as oil/water emulsions, various types of wetting agents, sterile solutions etc. Such carriers can be formulated by conventional methods and can be administered to the subject at an effective dose.

(18) The term parvovirus as used herein comprises wild-type or modified replication-competent derivatives thereof, as well as related viruses or vectors based on such viruses or derivatives. Suitable parvoviruses, derivatives, etc. as well as cells which can be used for actively producing said parvoviruses and which are useful for therapy, are readily determinable within the skill of the art based on the disclosure herein, without undue empirical effort.

(19) An effective dose refers to amounts of the active ingredients that are sufficient to affect the course and the severity of the disease, leading to the reduction or remission of such pathology. An effective dose useful for treating and/or preventing these diseases or disorders may be determined using methods known to one skilled in the art (see for example, Fingl et al., The Pharmocological Basis of Therapeutics, Goodman and Gilman, eds. Macmillan Publishing Co., New York, pp. 1-46 ((1975)).

(20) Additional pharmaceutically compatible carriers can include gels, bioasorbable matrix materials, implantation elements containing the therapeutic agent, or any other suitable vehicle, delivery or dispensing means or material(s).

(21) Administration of the compounds may be effected by different ways, e.g. by intravenous, intraperetoneal, subcutaneous, intramuscular, topical or intradermal administration. The route of administration, of course, depends on the kind of therapy and the kind of compounds contained in the pharmaceutical composition. A preferred route of administration is intravenous administration. The dosage regimen of the parvovirus and the HDACI is readily determinable within the skill of the art, by the attending physician based an patient data, observations and other clinical factors, including for example the patient's size, body surface area, age, sex, the particular parvovirus, HDACI etc. to be administered, the time and route of administration, the tumor type and characteristics, general health of the patient, and other drug therapies to which the patient is being subjected.

(22) If the parvovirus in the combination of HDACIs according to the invention comprises infectious virus particles with the ability to penetrate through the blood-brain barrier, treatment can be performed or at least initiated by intravenous injection of the virus, e.g., H1 virus. A preferred route of administration is intratumoral administeration.

(23) Since long-term intravenous treatment is susceptible to becoming inefficient as a result of the formation of neutralizing antibodies to the virus, different modes of administration can be adopted after an initial regimen intravenous viral administration, or such different administration techniques, e.g., intracranial or intratumoral virus administration, can be alternatively used throughout the entire course of parvoviral treatment.

(24) As another specific administration technique, the parvovirus (virus, vector and/or cell agent) can be administered to the patient from a source implanted in the patient. For example, a catheter, e.g., of silicone or other biocompatible material, can be connected to a small subcutaneous reservoir (Rickham reservoir) installed in the patient during tumor removal or by a separate procedure, to permit the parvovirus composition to be injected locally at various times without further surgical intervention. The parvovirus or derived vectors can also be injected into the tumor by stereotactic surgical techniques or by neuronavigation targeting techniques.

(25) Administration of the parvovirus can also be performed by continuous infusion of viral particles or fluids containing viral particles through implanted catheters at low flow rates using suitable pump systems, e.g., peristaltic infusion pumps or convection enhanced delivery (CED) pumps.

(26) A yet another method of administration of the parvovirus composition is from an implanted article constructed and arranged to dispense the parvovirus to the desired cancer tissue. For example, wafers can be employed that have been impregnated with the parvovirus, e.g., parvovirus H1, wherein the wafer is attached to the edges of the resection cavity at the conclusion of surgical tumor removal. Multiple wafers can be employed in such therapeutic intervention. Cells that actively produce the parvovirus, e.g., parvovirus H1, or H1 vectors, can be injected into the tumor or into the tumoral cavity after tumor removal.

(27) The combined therapy according to the invention is useful for the therapeutic treatment of cancers, e.g. prostate tumors, lung tumors, renal tumors, liver tumors, lymphoma, breast tumors, hepatoma or melanoma, in particular brain tumors, cervical carcinoma, or pancreatic carcinoma and can significantly improve the prognosis of said diseases. Parvovirus H1 infection effects killing of tumor cells but does not harm normal cells and such infection can, for example, be carried out by intracerebral use of a suitable parvovirus, e.g., parvovirus H1, or a related virus or vectors based on such viruses, to effect tumor-specific therapy without adverse neurological or other side effects.

(28) The present invention also relates to the use of (a) a parvovirus and (b) an HDACI for the preparation of (a) pharmaceutical composition(s) for the treatment of cancer wherein, preferably, (a) and (b) are sequentially (or separately) administered.

(29) In one preferred embodiment of the present invention, the combination of agents is utilized in the treatment of brain tumors such as glioma, medulloblastoma and meningioma, cervical carcinoma, or pancreatic carcinoma. Preferred gliomas are malignant human glioblastomas.

(30) In another preferred embodiment of the present invention, the parvovirus of the composition includes parvovirus H1 (H-1PV) or a related parvovirus such as LuIII, Mouse minute virus (MMV), Mouse parvovirus (MPV), Rat minute virus (RMV), Rat parvovirus (RPV) or Rat virus (RV).

(31) Patients treatable by the combination of agents according to the invention include humans as well as non-human animals. Examples of the latter include, without limitation, animals such as cows, sheep, pigs, horses, dogs, and cats.

(32) HDACIs useful for the purposes of the present invention include all HDACIs that are effective in inhibiting tumor growth. The administration of an HDACI can be accomplished in a variety of ways (see above) including systemically by the parenteral and enteral routes. Preferably, the parvovirus and the HDACI are administered as separate compounds. The preferred period of time between administration of the HDACI and the parvovirus is from 30 (administration of HDACI before the treatment with parvovirus) to 60 days after the treatment with parvovirus. Concomitant treatment with the two agents is also possible.

(33) Particular examples of HDCAIs suitable for the combined therapy include sodium butyrate (NaB), trichostatin A (TSA), suberoylanilide hydroxamic acid (SAHA) or valproic acid (VPA).

(34) The below examples explain the invention in more detail.

EXAMPLE 1

Materials and Methods

(35) (A) HDAC Inhibitors

(36) Sodium butyrate (NaB) and trichostatin A (TSA) were purchased from SIGMA-Aldrich (Munich, Germany), suberoylanilide hydroxamic acid (SAHA) and valproic acid (VPA) from Alexis Biochemicals (Enzo Life Science, Lorrach, Germany).

(37) (B) Lactate-Dehydrogenase Assay (LDH)

(38) Parvovirus cytotoxicity was measured by evaluating the release of lactate dehydrogenase in the medium using the CytoTox 96 kit from Promega (Mannheim, Germany). The day before infection, 2000 cells were seeded per well in 96-well plate in 50 l of culture medium. After 24 hours, cells were infected with 50 l of serum-free medium containing H-1PV or treated with HDAC inhibitors in 7 replicates per condition. The day of measurement, 3 out of 7 wells were incubated for 30 minutes with lysis buffer to estimate the maximum LDH release under conditions of 100% lysis. Afterwards, the plate was centrifuged and 50 l of supernatant were mixed with the substrate mix. The LDH enzyme catalyzes a colorimetric reaction that is indicative of the amount of enzyme released in the medium. After 30 minutes, the reaction was stopped and analyzed using the Elisa reader MultiScan at 492 nm.

(39) (C) Determination of the Sub-G1 Cell Population

(40) Virus infected or drug-treated cells were harvested from culture dishes with 0.05% Trypsin-EDTA solution, collected in a Falcon tube and then washed twice with PBS. Cells were then resuspended in 260 l of PBS, fixed with 700 l of cold 100% ethanol added drop wise under vortexing, and stored at 4 C. overnight. After two washes in PBS, the cell pellet was resuspended in a PBS solution containing 20 g/ml of RNase and 10 g/ml of propidium iodide (Sigma). Cell suspension was filtered and analyzed by FACSort flow cytometer (Becton-Dickinson). A minimum of 20,000 events were acquired and then analyzed with the CellQuest software (Becton-Dickinson) (San Jose, Calif.).

EXAMPLE 2

Parvovirus H-1PV and HDAC Inhibitors Act Synergistically in Killing Cervical Carcinoma Derived Cell Lines (HeLa, CaSki, SiHa) and Primary Tumor Cells

(41) It was found that sub-lethal doses of HDACIs, namely sodium butyrate (NaB), trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) increase PV cytotoxicity in a synergistic manner in cervical carcinoma derived HeLa (positive for HPV-18), CaSki and SiHa (positive for HPV-16) and primary cervical carcinoma cells.

(42) FIG. 1 shows the results obtained from LDH assays in HeLa and SiHa cells using various H-1PV multiplicities of infection (MOI=pfu/cell) in combination with the three HDACIs tested. Similar results were also obtained in CaSki cells (data not shown) and primary cervical cancer cells (CxCa) (FIG. 2) where a synergistic effect of up to 94%, resulting in approximately 80% cell death was achieved by combining SAHA (200 nM) with H-1PV (MOI=100 pfu/cell). Synergistic effects in killing the cancer cells above mentioned, were also observed using parvovirus in combination with another HDACI, namely VPA (data not shown).

(43) The synergistic effect in killing cancer cells between H-1PV and HDACIs was confirmed by flow cytometry in HeLa and CxCa, analyzing the fraction of cells containing less than 2N DNA content (sub-G1 cell population), as a marker of DNA fragmentation, a common feature of cells undergoing apoptosis. FIG. 3 shows the results obtained in HeLa cells. H-1PV infection is associated with an increase of the apoptotic sub-G1 cell population that is further enhanced by the addition of NaB (from 18% to 32%).

(44) FIG. 4 shows the results obtained in primary cervical cancer cells. H-1PV and HDAC inhibitors act synergistically in killing CxCa cells as demonstrated by the increase of apoptosis (from 26 to 37%). The conditions, in which H-1PV/HDACIs combinations give the highest synergistic effect in killing cervical carcinoma derived cells are summarized in Table 1.

(45) TABLE-US-00001 TABLE 1 Conditions under which H-1PV and HDACIs combinations show the highest synergistic effect in killing cervical cancer cells H-1PV + [NaB] H-1PV + [TSA] H-1PV + [SAHA] % synergy, % synergy, % synergy, Cell line MOI mM (% killed cells) MOI nM (% killed cells) MOI nM (% killed cells) HeLa 5 1 71 (97) 10 20 50 (80) 10 100 26 (60) SiHa 25 2 68 (80) 50 20 75 (83) 10 100 38 (57) Primary cervical 100 1 87 (85) 50 100 47 (52) 100 200 94 (72) cancer cells

EXAMPLE 3

Parvovirus H-1PV and HDAC Inhibitors Act Synergistically In Vitro (a) and In Vivo (b) in Killing Cervical Carcinoma Derived Cell Lines and Primary Tumor Cells

(46) (a) In Vitro Synergism.

(47) It was found that sub-lethal doses of HDACIs, namely Valproic acid (VPA), sodium butyrate (NaB), trichostatin A (TSA) and suberoylanilide hydroxamic acid (SAHA) potentiate H-1PV cytotoxicity in a synergistic manner in cervical carcinoma cell lines HeLa, positive for HPV-18, CaSki and SiHa, transformed by HPV-16 and low passage cervical carcinoma cell cultures.

(48) FIG. 5 shows the results obtained from LDH assays (which measure the release of lactate dehydrogenase in the medium, as an indicator of cell lysis) using H-1PV at various multiplicities of infection (MOI, expressed in plaque-forming units [pfu] per cell) in combination with 1 mM VPA. When applied singly, VPA did not have any significant effect on cellular growth at the concentration used, while the parvovirus killed all the cervical carcinoma cell cultures in a MOI-dependent fashion. The sub-lethal dose of VPA boosted parvovirus-induced cell killing in a synergistic manner, increasing by up to 50-100%. Confirmation of these results was obtained by flow cytometric determination of the apoptotic sub-G1 cell population (data not shown). Similar results were also obtained combining H-1PV with NaB, TSA or SaHa (Table 2).

(49) TABLE-US-00002 TABLE 2 Conditions under which H-1PV and HDACIs combinations show the highest synergistic effect in killing cervical cancer cells. Cell [NaB] + H-1PV [SAHA] + H-1PV [TSA] + H-1PV lines mM MOI Synergy * nM MOI Synergy * nM MOI Synergy * HeLa 1 1 78 (51) 50 3 63 (34) 50 3 46 (51) 1 3 68 (79) 50 5 45 (37) 50 5 37 (56) 1 5 71 (97) 50 10 27 (60) 50 10 45 (88) CaSki 0.5 2 43 (54) 100 5 43 (61) 50 2 75 (45) 0.5 5 48 (77) 100 10 39 (85) 50 5 77 (73) 0.5 10 49 (91) 100 20 49 (100) 50 10 82 (96) SiHa 2 10 94 (59) N.A. 100 10 46 (40) 2 25 48 (67) N.A. 100 25 92 (76) 2 50 47 (89) N.A. 100 50 89 (82) CxCa 1 10 50 (55) 200 10 105 (26) 100 10 107 (28) 1 25 61 (70) 200 25 90 (53) 100 25 77 (36) 1 50 111 (100) 200 50 95 (73) 100 50 47 (53) * Synergy is expressed as % increase in virus-induced killing. Number in brackets indicate percentages of killed cells. At the concentration used, the HDACIs had no or little effect on tumour cell growth when applied singly.
(b) In Vivo Synergism

(50) In order to validate the H-1PV/HDACIs synergism in vivo, a HeLa xenograft nude rat model was used. H-1PV alone was able to achieve the full regression of established tumours, when a high virus dose was administrated (2.510.sup.9 pfu/animal, fractioned in 4 intratumoral administration at weekly interval (data not shown). At a lower virus dose of 1.2510.sup.9 (pfu/animal, fractionated as above) tumour growth slowed down but non regression took place, indicating that a critical dose of the virus is required in order to have a therapeutic effect. At the concentration of 100 mg/kg, VPA failed to impair tumour growth which was comparable to mock-treated controls (rats were sacrificed when tumours reached the maximum tolerable size of 5 cm.sup.3). In contrast, tumour growth was strikingly reduced and eventually arrested at the size of 1-3.2 cm.sup.3, if animals injected with the lower viral dose were co-treated with VPA. Most remarkably, this stabilization was followed by a rapid regression leading to the complete disappearance of pre-existing tumours in all co-treated animals (FIG. 6). No loss of weight or other adverse side effects were documented in any of the treated animals. These results further strengthen the concept of combining H-1PV and HDACIs for the treatment of cervical carcinomas.

EXAMPLE 4

Parvovirus H-1PV and HDAC Inhibitors Act Synergistically in Killing Pancreatic Ductal Adenocarcinoma (PDAC) Derived Cell Lines (APC-1 and Capan-1)

(51) In Vitro Synergism.

(52) It was found that sub-lethal doses of HDACIs, namely Valproic acid (VPA) increase PV cytotoxicity in a synergistic manner in pancreatic ducal adenocarcinoma (PDAC) derived AP-1 and Capan-1 cell lines.

(53) FIG. 7 shows the results obtained from LDH assays in AP-1 and Capan-1 cell lines that have been described to be quite resistant to PV cytotoxicity (Dempe et al., 2010). Cells were seeded in 96 wells plate and then infected with various concentrations of H-1PV in the presence or absence of the HDACI Valproic acid (VPA). After 72 hours cell lysis was measured by LDH assay. As shown in FIG. 7, at the concentrations used for the experiments, single treatment with VPA had no effect on the growth of both cell lines. Consistent with previous results, H-1PV displayed only a limited cytotoxicity against these cultures, even after infection at high multiplicities. The cytotoxic activity of H-1PV was strikingly enhanced by VPA. These results further support combining HDACIs and H-1PV for the treatment of various tumour entities.

LIST OF REFERENCES

(54) Dempe S, Stroh-Dege A Y, Schwarz E, Rommelaere J, Dinsart C. (2010) SMAD4: a predictive marker of PDAC cell permissiveness for oncolytic infection with parvovirus H-1PV. Int J Cancer 126: 2914-27.