Targeted liposomal delivery of cGMP analogues

Abstract

The invention relates to means and methods of targeted drug delivery of therapeutic agent to and across the blood-ocular barrier. In particular, the invention relates to cyclic guanosine-3, 5-monophosphate analogs as therapeutic agent for treating retinal diseases. The cGMPSs targeted to the blood-ocular barrier by glutathione-based ligands that facilitate the specific binding to and enhanced internalization by glutathione transporters present on the blood-ocular barrier. The glutathione-based ligands are conjugated to nanocontainers such as liposomes encapsulating the cGMPSs.

Claims

1. A pharmaceutically acceptable nanocontainer comprising an agent for treating or diagnosing a pathology, condition or disorder associated with dysregulation of a cGMP-effected cellular target, wherein the target is at least one of a cGMP-dependent protein kinase (PKG), a hyperpolarization-activated cyclic nucleotide-gated (HCN) channel, a phosphodiesterase (PDE) and a cGMP-gated channel (CNGC), wherein the agent is a cyclic guanosine-3, 5-monophosphate analogue of the formula II: ##STR00014## wherein X.sup.2 is hydrogen, F, Cl, Br, I, CF3 or a NR.sup.9R.sup.10 or SR.sup.11 group, wherein R9 is hydrogen and both R.sup.10 and R.sup.11 are alkyl groups with a terminal NH.sub.2 or OH group, R.sup.8 is hydrogen, a (tri)alkylsilyl group or an acyl group, L is oxygen, sulphur, borano (BH.sub.3) or a further substituted borano group, and M is O(H), or L is O(H), and M is oxygen, sulphur, borano (BH.sub.3) or a further substituted borano group, Kat.sup.+ is Ca.sup.2+, and wherein the agent is encapsulated into the nanocontainer by remote loading.

2. The nanocontainer according to claim 1, wherein the nanocontainer is conjugated to a ligand for a glutathione transporter.

3. The nanocontainer according to claim 2, wherein the cyclic guanosine-3, 5-monophosphate analogue is the calcium salt of Rp-8-Br-PET-cGMPS.

4. The nanocontainer according to claim 2, wherein the ligand is selected from the group consisting of: glutathione, S-(p-bromobenzyl)glutathione, gamma-(L-gamma-azaglutamyl)-S-(p-bromobenzyl)-L-cysteinylglycin, S-butylglutathione, S-decylglutathione, glutathione reduced ethyl ester, glutathionesulfonic acid, S-hexylglutathione, S-lactoylglutathione, S-methylglutathione, S-(4-nitrobenzyl)glutathione, S-octylglutathione, S-propylglutathione, n-butanoyl gamma-glutamylcysteinylglycine, ethanoyl gamma-glutamylcysteinylglycine, hexanoyl gamma-glutamylcysteinylglycine, octanoyl gamma-glutamylcysteinylglycine, dodecanoyl gamma-glutamylcysteinylglycine, GSH monoisopropyl ester (N(N-L-glutamyl-L-cysteinyl)glycine 1-isopropyl ester sulfate monohydrate) and glutathione derivatives of the formula V: ##STR00015## wherein ZCH.sub.2 and YCH.sub.2, or ZO and YCO; R.sub.1 and R.sub.2 are independently selected from the group consisting of H, linear or branched alkyl (1-25C), aralkyl (6-26C), cycloalkyl (6-25C), heterocycles (6-20C), ethers or polyethers (3-25C), and where R.sub.1-R.sub.2 together have 2-20C atoms and form a macrocycle with the remainder of formula VI; R.sub.3 is selected from the group consisting of H and CH.sub.3; R.sub.4 is selected form the group consisting of 6-8C alkyl, benzyl, naphthyl and a therapeutically active cyclic guanosine-3, 5-monophosphorothioate; and, R.sub.5 is selected from the group consisting of H, phenyl, CH.sub.3- and CH.sub.2-phenyl; or, a pharmaceutically acceptable salt thereof.

5. The nanocontainer according to claim 4, wherein R.sub.3 is H, R.sub.4 is benzyl, and R.sub.5 is phenyl.

6. The nanocontainer according to claim 2, wherein the nanocontainer is a liposome encapsulating the therapeutic or diagnostic agent, and wherein the ligand for a glutathione transporter is conjugated to the liposome through a bifunctional conjugation agent comprising a vitamin E derivative or a phospholipid bonded to one end of the conjugation agent and the ligand for a glutathione transporter bonded to the other end of the conjugation agent.

7. The nanocontainer according to claim 6, wherein the conjugation agent is polyethylene glycol having polymerization number (n) of between 6-210.

8. The nanocontainer according to claim 7, wherein the polyethylene glycol has a molecular weight between 1,000 and 5,000 Da.

9. The nanocontainer according to claim 6, wherein the conjugation agent is obtainable by reacting distearoylphosphatidylethanolamine-polyethylene glycol-maleimide (DSPE-PEG-MAL) with a ligand for a glutathione receptor having a maleimide-reactive thiol group.

10. The nanocontainer according to claim 9, wherein the DSPE-PEG-MAL has a molecular weight of about 2,000 Da.

11. The nanocontainer according to claim 2, wherein the ligand for a glutathione transporter is glutathione.

12. A pharmaceutical composition comprising a nanocontainer according to claim 1 and pharmaceutically acceptable carrier.

13. A method of treating at least one of: (a) retinitis pigmentosa or another a hereditary disease of the retina; (b) secondary pigmentary retinal degeneration as a results of a metabolic or neurodegenerative disease, a syndrome or an eye disease; (c) diseases of the retina comprising diabetic retinopathy, age related macular degeneration, macular Hole/Pucker, retinoblastoma, retinal detachment and river blindness, the method comprising administering to a subject in need thereof a nanocontainer according to claim 1.

14. The method according to claim 13, wherein the nanocontainer is administered systemically or locally.

15. The method according to claim 14, wherein the nanocontainer is administered by at least one of (a) injection or infusion by at least one of intravitreal, intravenous, intraperitoneal, and intraarterial routes; and (b) topical or ocular application.

16. The method according to claim 13, wherein the nanocontainer is administered in doses of between 0.1 and 1000 mg/kg once per 1 or 2 days.

17. The method according to claim 13, wherein the nanocontainer is administered intravitreally in doses of between 0.0005 and 0.02 mg/kg once per two weeks or once per six weeks.

Description

BRIEF DESCRIPTION OF THE FIGURES

(1) FIGS. 1A and 1B. In vitro protective effects of DF003 (Rp-8-Br-PET-cGMPS) and LP-DF003 (Rp-8-Br-PET-cGMPS encapsulated in GSH-conjugated liposomes prepared as described in Example 1). DF003 significantly reduced the death of rd1 photoreceptor-like cells, at concentrations as low as 100 nM. This effect was increased by LP-DF003, which was still effective when it was used to yield a dose that corresponded to 10 nM of DF003. At the same time wild-type (wt) photoreceptor-like cells were not affected by either DF003 or LP-DF003, even at the highest concentration used (50 M). B) Similarly, DF003 reduced photoreceptor death in organotypic rd1 retinal explant cultures, while it showed no signs of toxicity in wt explant cultures.

(2) FIGS. 2A and 2B. LP-DF003 protects photoreceptors in three different retinitis pigmentosa animal models. Photoreceptor survival was assessed in three different in vivo retinitis pigmentosa mouse models, at post-natal day (PN) PN14 in rd1 mice; and at PN30 in rd2 and rd10 mice. Green bars represent the wild-type (wt) situation; red bars represent the untreated mutant situation. A) rd1 animals treated with free DF003 did not show any improvement on photoreceptor survival when compared to untreated rd1. In contrast, LP-DF003 treatment significantly preserved rd1 photoreceptors at PN14. B) At PN30 retinal degeneration in rd2 mice has caused the loss of approx. 15% of photoreceptors, while in rd10 retina at the same age around 80% are lost. In both rd2 and rd10 animals, treatment with LP-DF003 significantly increased the number of surviving photoreceptors.

(3) FIGS. 3A-3C. LP-DF003 preserves photoreceptor viability and function in rd10 animals in vivo. In mice, retinitis pigmentosa progresses from the centre (optic nerve=0) to the periphery (90). A) LP-DF003 rescued rd10 photoreceptors (ONL) in the dorsal parts of the peripheral retina, indicating slower disease progression. B) Representative electroretinographic (ERG) responses in untreated (red) and LP-DF003 treated (orange) rd10 animals. Adult wild-type traces (green) are shown for comparison. The bar graph shows that average (n=7) b-wave ERG amplitudes are 4-5 fold larger in treated rd10 animals.

(4) FIG. 4. Pharmacokinetics of DF003 and LP-DF003. A pharmacokinetic study in adult rats revealed a strong extension of the in vivo half-life of LP-DF003 vs. free DF003. While free DF003 had an in vivo half-life of around 15 min, LP-DF003 displayed a half-life time of approximately 24 hrs.

(5) FIGS. 5A-5D. SLO imaging in vivo demonstrates successful delivery to the retina. Mice injected at postnatal day (PN) 10 with fluorescently labelled liposomal drug delivery system were analysed in vivo at PN14 and PN20 using scanning laser ophthalmoscopy (SLO). Topical application (not shown), and intravitreal (a) and subtenon (b) injection did not show significant retinal uptake of label. However, intravenous (c) and intraperitoneal (d) injection, both, resulted in a strong fluorescent labelling of retinal blood vessels and neuroretina at PN14, i.e. 4 days after application. At PN20 most of the label had disappeared, although after intraperitoneal injection defined retinal patches still showed some fluorescence.

EXAMPLES

(6) 1. Materials and Methods

(7) 1.1 Production of GSH-Conjugated Liposomes Encapsulating cGMPS

(8) First, micelles were prepared by mixing (molar ratio 1:1.5) DSPE-PEG2000-maleimide (NOF, Grobbendonk, Belgium, 916 mg in 36.72 mL of DI water) with glutathione (Sigma-Aldrich, Zwijndrecht, the Netherlands, 144 mg in 4.42 mL of DI water) at room temperature for 2 h. Next, micelles were added to calcium acetate hydrate (4094 mg in 57.36 mL of DI water; final concentration 200 mM) and kept at 60 C. for 30 minutes.

(9) 2808 mg of HSPC (Hydrogenated Soy Phosphatidylcholine; final concentration 28 mM) and 912 mg of cholesterol (final concentration 18.6 mM) were dissolved in 30.96 mL ethanol in a serum bottle, mixed with the micelles while stirring and incubated in a water bath for 30 min at 60 C. Finally, the liposomes were extruded using 0.2/0.2 m PC membrane (2 times), 0.2/0.1 m PC membrane (2 times) and 0.1/0.1 m PC membrane (2 times) at 60 C. and stored at 4 C. The size of liposomes was measured with 10 L of the liposomal suspension diluted in 1 ml PBS by the dynamic light scattering method (Zetasizer Nano ZS, Malvern, Worcestershire, UK). The average size of the liposome batches was between 100 and 120 nm, PdI<0.1. After size was measured the calcium acetate liposomes were purified from non-encapsulated calcium acetate hydrate using dialysis system TFF (Millipore Cogent Scale and Millipore Pellicon cassette 50 cm.sup.2). The liposomes were dialyzed against 7 volumes of saline (0.9% NaCl). The batch was concentrated back to start volume 120 mL using dialysis with a TFF system and analyzed for lipid content and size.

(10) LP-DF003 was generated by remote loading of the calcium acetate GSH-PEG-liposomes with DF003 (Rp-8-Br-PET-cGMPS) at a drug/phospholipid molar ratio 0.3. For this 1 volume of DF003 dissolved in MilliQ (40 mg/mL) was mixed with 9 volumes of liposomes (HSPC 20 mg/mL, both pre-warmed at 60 C.) and incubated at 60 C. for 45 min. Subsequently, the batch was stored at 4 C., purified and analyzed. Purification was done by dialysis using a TFF system. The LP-DF003 liposomes were dialyzed against 10 volumes of saline, concentrated to a DF003 concentration of 3 mg/mL and sterile filtered with 0.2 m filter (Corning sterile syringe filter) and stored at 4 C. Encapsulated DF003, i.e. LP-DF003, and other liposome constituents were analyzed by HPLC.

(11) For encapsulation Sp-8-Br-PET-cGMPS and 8-Br-PET-cGMP by remote loading essentially the same procedure is applied as above for encapsulation of LP-DF003.

(12) Encapsulation by remote loading of DF001 (Rp-8-Br-cGMPS), DF002 (Rp-8-pCPT-cGMPS) and Rp-8-pCPT-PET-cGMPS was also tested, using several experimental conditions (including drug-lipid ratio, extraliposomal pH, intraliposomal pH, and intraliposomal calcium acetate concentration) were screened to facilitate and sustain the encapsulation of these drugs. In contrast to DF003, encapsulation by remote loading of DF001, DF002 and Rp-8-pCPT-PET-cGMPS was not possible.

(13) Rp-8-Br-cGMPS, Rp-8-pCPT-cGMPS, Rp-8-Br-PET-cGMPS (DF003) and Rp-8-pCPT-PET-cGMPS were obtained from BIOLOG Life Science Institute GmbH, Bremen, Germany.

(14) 1.2 Animals for In Vitro Retinal Explant Culture and for In Vivo Studies

(15) Animals for preparation of primary retinal cell cultures were kept at CSSI (Centro Servizi Stabulario Interdipartimentale) of University of Modena and Reggio Emilia. The protocol was approved by the Ethical Committee of University of Modena and Reggio Emilia (Prot. N. 106 22 Nov. 2012) and by Italian Ministero della Salute. Animals for in vitro retinal explant studies were kept at the Lund University department for clinical sciences. Here, we used the rd1 and rd2 retinitis pigmentosa model mice with corresponding wild-type (wt) controls. Animals were kept under standard white cyclic lighting, with ad libitum access to food and water, and were used irrespective of gender. All procedures were performed in accordance with the Swedish animal care and ethics committees. Efforts were made to keep the number of animals used and their suffering to a minimum.

(16) Animals for in vivo studies were kept in the Tubingen Institute for Ophthalmic Research internal animal housing facility, under standard white cyclic lighting, had free access to food and water, and were used irrespective of gender. C3H rd1/rd1 (rd1) and control C3H wild-type (wt) mice were for initial in vivo testing. After successful testing in rd1 animals, the in vivo testing was extended to further RD animal models for cross-model validation. These additional animal models were: C3H rd2/rd2 (rd2 or rds), C57Bl6J rd10/rd10 (rd10), C57Bl6J cpfl1/cpfl1 (cpfl1) mice, as well as Rho P23H rats. All in vivo procedures were performed in accordance with the local ethics committee at Tubingen University ( 4 registrations from 29-04-10; 30-06-10; 11-03-11; animal permit AK5/12), and the ARVO statement for the use of animals in ophthalmic and visual research.

(17) 1.3 Primary Retinal Cell Culture Preparation, Differentiation and Treatment

(18) Different doses of DF003 and LP-DF003 were tested in vitro on a primary culture of retinal cells derived from the rd1 mouse model. About 20-30% of the primary cells can be differentiated into rod photoreceptors (Demontis et al. 2012, PLoS One. 7, e33338; Giordano et al. 2007, Mol. Vis. 13, 1842-1850). Retinal stem cells from adult wt and rd1 mice were isolated from the ciliary epithelium after treatment with 2 mg/ml dispase (20 min) followed by 1.33 mg/ml trypsin 0.67 mg/ml, hyaluronidase and 0.13 mg/ml kynurenic acid (10 min) and cultured for a week in serum free medium containing 20 ng/ml basic FGF, 2 g/ml heparin, 0.6% glucose, N2 hormone mix in DMEM-F12 to form neurospheres (Giordano et al., 2007, supra). Retinal neurospheres were then plated on glass slides coated with extracellular matrix (ECM, Sigma) in DMEM-F12 supplemented with 20 ng/ml FGF for 4 days. Cells were allowed to differentiate in DMEM-F12 supplemented with 1% FBS. rd1 differentiated retinal cells activated a cell death program at the 11.sup.th day of differentiation as previously published (Sanges et al. 2006, Proc. Natl. Acad. Sci. U.S.A 103, 17366-17371). Cells were exposed to different doses of LP-DF003 at day 10 of differentiation (one day before activation of cell death pathways). 16 hours after treatment with LP-DF003 cells were fixed with 4% paraformaldehyde for 10 min at room temperature. Cell death was evaluated by staining of cells for 2 minutes with 2 M Ethidium homodimer and counterstaining of nuclei with DAPI (4,6-diamidino-2-phenylindole, Sigma). Slides were mounted with mowioll 4-88 (Sigma) and analysed at a Zeiss Axioskop 40 fluorescence microscope. Ethidium homodimer positive cells were counted in each slide and expressed as percentages of the total number of cells (DAPI stained) per slide. Paired Student's t-test analysis compared data derived from at least three different untreated and at least three different treated rd1 retina cells.

(19) 1.4 Organotypic Retinal Explant Culture

(20) For biochemical analyses and comparisons between models and wt tissues, we typically use material from ages corresponding to the onset of retinal degeneration. Retinal tissue is obtained from post-natal day 5 (PN5) animals, from which, after sacrifice, the eyes are enucleated and retinae cultured as retinal explants. In brief, the retina with the retinal pigment epithelium (RPE) still attached is isolated and subsequently transferred to Millicell culture dish filter inserts (Millipore AB, Solna, Sweden; PIHA03050), with the RPE layer facing the culturing membrane. The explants are then incubated in custom made R16 nutrient medium at 37 C. The nutrient medium has a volume of 1.5 ml per dish, which is replaced with fresh medium usually every second day (unless the exact culturing paradigm requires otherwise) during the culturing period.

(21) PN5 explants were allowed to adjust to culture conditions for two days in vitro (DIV), after which they were subjected to treatments of interest. At this point the treatment paradigm consisted of addition of medium with test compound every second day for four days reaching the equivalent to PN11 (labelled as short term treatment: PN5+2 DIV+4 DIV) or to PN19 (long term treatment: PN5+2 DIV+12 DIV).

(22) At the end of the explant culturing period, the specimens were fixed in 4% paraformaldehyde in a phosphate-buffered salt solution for about 2 h in 4 C. The thus fixed eyes were cryoprotected in Sorensen's sucrose buffer, histological sectioning was performed using a cryotome, and 12 m cryosections collected on microscope slides.

(23) 1.5 Drug Testing on In Vivo RD Animal Models

(24) Before treatment with drug or drug/DDS combinations, the animals were anesthetized with diethyl-ether. For systemic treatment, 0.9% NaCl solution containing liposomal DDS/drug formulation were injected either into the tail vein (caudal vein; intravenous; i.v.; 50 l) or into the peritoneum (intraperitoneal; i.p.; 200 l) of the anesthetized animal. In rd10 mice, a local intravitreal (IVT) treatment was also tested. Here, the animals received a 0.5 l injection into the vitreous body of one eye, while the other eye was kept as untreated, contralateral control. For both i.p. and IVT treatments a liposomal formulation not containing DF003 (i.e. empty liposomes) was used as an additional control.

(25) The in vivo treatment was performed initially on rd1 animals, but later extended to other animal models (rd2, rd10, cpfl1, Rho P23H). Because of the different onset and progression of retinal degeneration in the different RD models, the treatment paradigms had to be adapted to each model. For details on these treatment paradigms see Table 1.

(26) At various post-treatment time-points (see Table 1), in vivo optic coherence tomography (OCT) and scanning laser ophthalmoscopy (SLO) analysis was used for direct, non-invasive imaging of retinal morphology (OCT) and of fluorescently labelled drugs (SLO) or drug/DDS combinations to determine the distribution and uptake of drug in the retina. In addition, retinal function was assessed using electroretinographic (ERG) recordings. After non-invasive in vivo examinations, and between 1 to 12 days after treatment, experimental animals were killed by carbon dioxide asphyxiation. The eyes were immediately enucleated, fixed for 2 h in 4% PFA and prepared for cryosectioning or whole-mount preparation.

(27) TABLE-US-00001 TABLE 1 In vivo treatment paradigms for five different RD models Animal model, Treatment Treatment In vivo Treatment Species start intervals/dosis analysis end rd1 mouse PN10 Once per day/ PN14, 18, (30) PN17 (i.v. + i.p. 200 l on the first day) rd2 mouse PN14 Every 2.sup.nd day/ PN18, 30, 60 PN59 200 l rd10 mouse PN14 Once per day/ PN18, 24, 30 PN29 200 l rd10 mouse PN14 PN14, 16, 18, PN30 PN26 IVT 22, 26/0.5 l cpfl1 mouse PN14 Once per day/ PN24, 30 PN29 200 l Rho P23H PN14 Every 4.sup.th day/ PN30, 60, 120 PN118 rat 400-3200 l
1.6 Quantification of Photoreceptor Cell Death and Survival

(28) The read-outs for the in vitro and in vivo treatment experiments consisted of quantification of photoreceptor cells stained positive for the cell death marker TUNEL and/or counting of surviving photoreceptor rows, as seen in standard histological tissue stains. In both cases the results were captured by means of a microscope and digital camera, analysed manually or semi-automatically, and this was then followed by calculations for statistical significance of the recorded data in principle as published previously (Arango-Gonzalez et al. 2014, PLoS One. 9, e112142).

(29) 2. Results

(30) 2.1 In Vitro and In Vivo Protection of Photoreceptors by LP-DF003

(31) Over 200 novel cGMP analogues generated, of which 140 were tested for their capacity to bind PKG in cell free assays (Zegzouti et al. 2009, Assay. Drug Dev. Technol. 7, 560-572). Of these, 33 compounds exhibiting strong PKG binding, were selected for further in vitro analysis in the 661W cell line and in photoreceptor-like cell cultures derived from retinitis pigmentosa mutant mice (Sanges et al. 2006, supra). This cell-based screening assay identified 11 compounds that could reduce cell death caused by retinitis pigmentosa mutations and thus showed photoreceptor protective activity. These 11 compounds were then further tested in organotypic retinal explant cultures (Sahaboglu et al. 2013, Cell Death & Disease 4) derived from either wild-type, rd1 (Sanyal and Bal 1973, Z. Anat. Entwicklungsgesch. 142, 219-238) or rd2 mice (Sanyal and Jansen 1981, Neurosci. Lett. 21, 23-26). Retinal tissue cultures narrowed down the number of cGMP analogues with promising neuroprotective effects to 4 compounds, which were found to significantly reduce cell death of rd1 and rd2 photoreceptors in vitro.

(32) These 4 compounds were then tested in vivo in rd1 mice, and one of these in combination with the liposomal (LP) delivery system. LP-DF003 showed the most pronounced protective effects in rd1 animals and was then subjected to tests in two other in vivo mouse models for retinitis pigmentosa (rd2 and rd10 mice). Another long-term study (4 months) was performed in a fourth retinitis pigmentosa model, the P23H rat (data not shown).

(33) In the different test systems DF003 yielded the following results: DF003 preserved the viability of diseased rd1 photoreceptors in cell and organotypic retinal tissue cultures (FIG. 1 A, B). In both systems, wild-type (wt) photoreceptors were not affected by DF003 treatment indicating that it was not toxic to these cells up to the concentration of 50 M. LP-DF003 showed improved protective effects when compared to DF003 at lowed concentrations in photoreceptor-like cell cultures derived from retinitis pigmentosa mutant mice (FIG. 1A)

(34) In organotypic retinal explants, no evidence of DF003 toxicity was found in the inner retina (data not shown). DF003 also prevented photoreceptor death in retinal explants of two other models, the more slowly degenerating rd10 model, which also has a Pde6b mutation, but at a different site than the rd1 model, as well as the even slower degenerating rd2 mouse model in spite of the very different mutation the rd2 mouse carries (data not shown, but see FIG. 2 FIGS. 2A and 2B for the in vivo data of rd10 and rd2). See also below for more details on these models. Similar observations were made for Sp-8-Br-PET-cGMPS.

(35) The bioavailability of DF003 in vivo was dramatically improved when it was used in its liposomal formulation LP-DF003 (cf. section 2.3; FIG. 4). Also the bioavailability of Sp-8-Br-PET-cGMPS improves when encapsulated into liposomes.

(36) To assess the effects of LP-DF003 in vivo, we used three different retinitis pigmentosa animal models carrying genetic defects homologous to human retinitis pigmentosa mutations. The rd1 mouse is an animal model for retinitis pigmentosa with a loss of function of the Pde6b gene, leading to a rapid cell death of rod photoreceptors until post-natal (PN) day 18 (Sanyal and Bal 1973, vide supra). At PN14 the rd1 retina has lost more than 50% of its photoreceptor rows, when compared to the wt (FIG. 2A). While systemic administration of DF003 via intraperitoneal injection (50 mg/kg once per day) had no effect on rd1 photoreceptor survival, treatment with LP-DF003 at the same concentration significantly increased rd1 photoreceptor viability.

(37) The rd2 (rds) mouse is another model for retinitis pigmentosa carrying a mutation in the Prph2 gene that leads to a relatively slow loss of rod and cone photoreceptors in the first three post-natal months (Sanyal and Jansen 1981, vide supra). At PN30 about 15% of rd2 photoreceptors are lost. Already at this age rd2 animals treated with LP-DF003 (50 mg/kg every second day) exhibited a significant increase in the number of surviving photoreceptor rows (FIG. 2B).

(38) The third animal model used was the rd10 mouse bearing a point mutation in the Pde6b gene. Here, degeneration is slower when compared to the rd1 mutant retina and the loss of rod photoreceptors starts at PN18 with around 80% lost at PN30. Also in rd10 animals, systemic administration of LP-DF003 via intraperitoneal injection (i.p.; 50 mg/kg once per day) significantly increased the survival of photoreceptors at PN30 (FIG. 2B). The same i.p. treatment of rd10 mice with control liposomes not containing DF003 (empty liposomes) did not yield any significant differences from untreated rd10 animals.

(39) The data shown in FIGS. 2A and 2B confirmed that overall photoreceptor survival was improved by LP-DF003 treatment. Since in rodent retinitis pigmentosa models the degeneration of photoreceptors progresses from the centre to the periphery, the protective effect was more pronounced in the periphery. This was assessed in so called Spider diagrams, which show the amount of surviving photoreceptors as a function of the eccentricity from the optic nerve, i.e. the centre of the retina. Thus, in the retinal periphery of LP-DF003 treated rd10 animals there were about two times more surviving photoreceptor than in untreated counterparts (FIG. 3A).

(40) More importantly, i.p. LP-DF003 strongly improved the in vivo functionality of the retina as assessed by ERG recording in rd10 animals. In ERG the negative deflection of the electric response to light, the so called a-wave, reflects the primary response of the photoreceptors. The subsequent positive deflection, the so-called b-wave, corresponds to the response of the inner retina and the activation of second order neurons. While adult wt animals display ERG responses ranging from 350 V (a-wave) to 600 V (b-wave), the ERG of untreated rd10 animals is almost extinguished at PN30 (maximal b-wave response35 V)(FIG. 3B). In contrast, i.p. LP-DF003 strongly and highly significantly improved rd10 ERG b-wave responses (180 V), corresponding to a 4-5 fold improvement of retinal function.

(41) Similar results were obtained when rd10 animal were treated with LP-DF003 via intravitreal injection (IVT). The injected eyes showed a strong increase in the numbers of surviving photoreceptor rows (treated: 4.660.45 SEM, contralateral: 1.570.17 SEM, n=8, p=0.0002). This was reflected by a corresponding increase in functional ERG responses, which at PN30 showed a highly significant increase to 250 V in the treated eyes.

(42) Overall the results of LP-DF003 treatment in the different retinitis pigmentosa animal models are highly encouraging and strongly highlight LP-DF003's potential for the development of a mutation-independent treatment for several different forms of human retinitis pigmentosa.

(43) When the in vivo effects of Sp-8-Br-PET-cGMPS encapsulated into liposomes are compared to ordinary Sp-8-Br-PET-cGMPS, in the above animal models, a similar improvement in biological potential by encapsulation of Sp-8-Br-PET-cGMPS is observed as found for LP-DF003 compared to DF003.

(44) 2.2 Pharmacology

(45) The active compound DF003 is an analogue of cGMP, blocking PKG activity with high specificity. This is particularly true for the PKG1 and PKG1 isoforms, while PKG2 is less well inhibited. The inhibitory constants for the two other potential targets CNGC and protein kinase A (PKA) isoforms 1 and 2 are 2-3 log units higher (Table 2) than those for PKG isoforms. cGMP and its analogues are targeting PKG and CNGC but could potentially also interfere with PKA, PDE or even HCN channel activity. The table gives the inhibitory constants (K.sub.i) of DF003 for these targets and (where available) the IC.sub.50 values.

(46) TABLE-US-00002 TABLE 2 Inhibitory constants for DF003 Target PKG1 PKG1 PKG2 CNGC PKA1 PKA2 K.sub.i 0.035 M 0.03 M 0.45 M n.a. >50 M 11 M IC.sub.50 0.9 M 25 M Target PDE1B PDE2 PDE4 PDE5 PDE10 K.sub.i 2.5 M 0.8 M 8.1 M 4.1 M 5.0 M
2.3 Pharmacokinetics

(47) A pharmacokinetic study was performed in adult rats (3 months old) that received a single injection of either free DF003 or LP-DF003 at an initial dose of 20 mg/kg. Results are shown in FIG. 4. While the free DF003 was very rapidly cleared away from the blood-stream (estimated half-life: 10-15 min), high levels of DF003 remained within the blood stream when LP-DF003 was administered. Here, the estimated half-life was 24 h, corresponding to a 90 to 100-fold extension of half-life, approximately.

(48) To ensure an optimal delivery of liposomes (e.g. containing DF003) to the retina, in both mice and rats a variety of application paradigms were tested for compounds encapsulated in the liposomal drug delivery system. These included topical application (eye drops), intravitreal injection into the eye, subtenon injection into the Tenon capsule surrounding the eye, intraperitoneal injection, and intravenous injection. The use of fluorescent tracer compounds within the liposomal delivery system made it possible to directly track compound delivery using scanning laser ophthalmoscopy (SLO; FIGS. 5A-5D). In mice and rats an intravenously applied fluorescent tracer (e.g. fluorescein) is otherwise cleared from the blood stream within a few hours.

(49) Remarkably, direct applications to or into the eye resulted in no or almost no drug delivery to the retina, presumably due to the strong adhesion of the liposomes to non-retinal ocular structures (e.g. the vitreous). In contrast, systemic administration of liposomes by both intravenous and intraperitoneal injection resulted in a strong compound uptake in the retina, something that may be explained on the one hand by the prolonged circulation in the blood stream (cf. FIG. 4) and on the other hand by a facilitation of targeted transcytosis across the blood ocular barrier into the neuroretina. In mice, with liposomal formulation, the fluorescent tracer could be directly visualized in the retina via SLO for at least 4 days after a single intraperitoneal injection (FIGS. 5A-5D); in rats (not shown) the tracer was detectable for at least 10 days post injection.

(50) 2.4. Toxicology

(51) Mice and rats treated with LP-DF003 (i.p.) for a duration of up to two months, as well as their untreated controls, were routinely examined in vivo and post mortem, without any macroscopic evidence for toxic drug effects.

(52) Treated animals in vivo showed no alterations in behaviour (e.g. apathy, hunched, kyphotic posture), in the appearance of fur (e.g. hair loss, oily fur), or their skin (e.g. discolorations, haemorrhages). In vivo eye examinations found no abnormalities (e.g. lens opacity, cataract), while functional ERG testing revealed better performance in LP-DF003 treated animals, compared to untreated controls. Importantly, treated and untreated animals showed normal weight gains during their first two post-natal months and were generally undistinguishable from each other (data not shown). Likewise, macroscopic post mortem examination of internal organs (heart, liver, lungs, kidney, brain) revealed no abnormalities (organ size and form, coloration/perfusion) in LP-DF003 treated mice and rats. When mice were treated with the highest dose of LP-DF003 (200 l i.p., every day), the spleen of some animals appeared bigger than in controls, a phenomenon that may be related to the administration of lipids. While this phenomenon did not seem to negatively affect the animals, it will be further evaluated and the lipid concentration in the drug formulation may be adapted accordingly.

(53) Taken together and based upon these preliminary data, there is no evidence of any apparent and strong toxicological effects in these animals under the experimental conditions used. In particular, if in the human application an intravitreal injection is envisaged, the doses of LP-DF003 to be applied would be at least 400-1000 fold lower than what was used in the mouse and rat experiments.