PHARMACEUTICAL NANOSUSPENSION FOR THE THERAPY OF HIV INFECTION

Abstract

The present invention relates to a pharmaceutical composition (nanosuspension) for long-acting injectable (LAI) drug for long-term supportive therapy of HIV/AIDS.

A pharmaceutical nanosuspension to be used as an injectable drug for long-term supportive therapy of HIV infection comprising a composition containing, as an active ingredient, a compound of general formula 1 in a crystalline or polycrystalline form

##STR00001## wherein is C.sub.2H.sub.5CON.sup.Na.sup.+, NH.sub.2.

Claims

1. A pharmaceutical nanosuspension as an injectable drug for long-term supportive therapy of HIV infection including a composition comprising, as an active ingredient, a compound of general formula 1 in a crystalline or polycrystalline form ##STR00004## wherein R is C.sub.2H.sub.5CON.sup.Na.sup.+, NH.sub.2.

2. The pharmaceutical nanosuspension according to claim 1 comprising a composition containing, as an active ingredient, a compound of formula 1b ##STR00005##

3. The pharmaceutical nanosuspension according to claim 1 with particle size varying from 200 nm to 900 nm.

4. A composition in the form of lyophilisate, said lyophilisate resulting from freeze drying of a nanosuspension with particle size varying from 200 nm to 900 nm and comprising, as an active ingredient, a compound of general formula 1 in a crystalline or polycrystalline form and in a therapeutically effective amount, optionally a pharmaceutically acceptable filler, additive, or diluent.

5. The composition according to claim 4 comprising a compound of formula 1b.

6. The composition according to claim 4 comprising a solubilizer (for example, poloxamer P338) and detergent (for example, mannitol or saccharose).

7. The composition according to claims 4-6 for the production of a pharmaceutical nanosuspension.

8. A pharmaceutical nanosuspension to be used as an injectable drug for long-term supportive therapy of HIV infection comprising the composition according to claims 4-6, a phosphate buffered saline (PBS), and water for injection.

9. A method for producing a composition according to claims 4-6 involving rotary grinding using zircon sand in an aqueous solution of the poloxamer of a compound of general formula 1 in a crystalline or polycrystalline form, solubilizer and detergent, followed by separation of zircon sand and lyophilization of the resulting suspension.

10. A method for producing a pharmaceutical nanosuspension by mixing the composition according to claims 4-6, phosphate buffered saline (PBS) having pH=6.8, and water for injection.

Description

[0034] This invention is illustrated with drawings.

[0035] FIG. 1. Particle size measurement curves for three samples following 24-hour grinding with saccharose.

[0036] FIG. 2. Particle size measurement curves for three samples following 24-hour grinding with mannitol.

[0037] FIG. 3. Particle size measurement curves for three samples following 24-hour grinding and 20-hour settling (saccharose).

[0038] FIG. 4. Particle size measurement curves for three samples following 24-hour grinding and 20-hour settling (mannitol).

[0039] FIG. 5. Particle distribution measurement curves for three samples from Lot 740-1-101.1 (saccharose) following lyophilization and resuspending.

[0040] FIG. 6. Particle distribution measurement curves for three samples from Lot 740-1-101.2 (mannitol) following lyophilization and resuspending.

[0041] FIG. 7. Concentration-time curves VM-1500A in canine plasma for single-dose SC and IM administration of pharmaceutical nanosuspensions VM-1500-LAI and VM-1500A-LAI (10 mg/kg).

PREFERRED EMBODIMENT

[0042] The present invention will now be described in terms of certain embodiments which are not intended to limit its scope. On the contrary, the present invention covers all alternatives, modifications, and equivalents that can be included within the scope of the claims. Thus, the following examples, which include specific embodiments, will illustrate this invention without limiting it.

Example 1. Method for Producing a Lyophilized Nanocomposition

[0043] Zircon sand (150 ml) (grinding media: 0.5 mm YTZ Zirconia Grinding and Dispersion Media; Tosoh USA, Inc.) and a solution of poloxamer P338 (10.0 g) and saccharose or mannitol (11.6 g) in 400 ml of PBS (pH=7.4) were placed into a jar. The resulting solution (150 ml) was placed into a jar, and zircon sand (150 ml) (grinding media: 0.5 mm YTZ Zirconia Grinding and Dispersion Media; Tosoh USA, Inc.) and compound VM11500A (15.07 g) were added. The jar was placed on a mill (U.S. Stoneware 700 Series jar mill), where the mixture was ground for 24 hours at 104 rev/min. The end of the process was checked by particle distribution analyses on the Malvern Zetasizer Nano ZS instrument. Upon completion of the process, the rotation was stopped and the mixture was allowed to stand for 16-20 hours at 2-8 C. to let the zircon sand settle. The suspension was filtered through a glass filter (filtration pore diameter: 5-15 m) and analyzed for concentration of the compound VM1500A (92-97% from nominal) to afford a Hananosuspension, which was aseptically poured into 5-ml sterile glass vials (2 ml each), frozen, and lyophilized to afford a lyophilized nanocomposition with characteristics given in Tables 1 and 2 and FIGS. 1, 2, 3, and 4.

TABLE-US-00001 TABLE 1 Particle size in the nanosuspension after grinding for 24 h (the mean of three measurements) Sample Particle Sample volume, l size, nm PDI* Quality Lot No. 740-1-101.1 (saccharose) 30 442.3 0.428 good Lot No. 740-1-101.1 (saccharose) 10 357.7 0.283 good Lot No. 740-1-101.2 (mannitol) 30 395.6 0.31 good Lot No. 740-1-101.2 (mannitol) 10 552.4 0.487 good *PDI is here and elsewhere a dispersion index, which characterizes the uniformity of particles in the mixture.

TABLE-US-00002 TABLE 2 Particle size in the nanosuspension after grinding for 24 h and settling for 20 h (the mean of three measurements) Sample Particle Sample volume, l size, nm PDI Quality Lot No. 740-1-101.1 (saccharose) 30 402.9 0.367 good Lot No. 740-1-101.1 (saccharose) 10 362.1 0.268 good Lot No. 740-1-101.2 (mannitol) 30 403.5 0.28 good Lot No. 740-1-101.2 (mannitol) 10 381.3 0.286 good

Example 2. Method for Producing a Pharmaceutical Nanosuspension

[0044] To the nanosuspension obtained in Example 1 was added a pre-prepared sterile PBS solution (pH=6.8) based on 2.2 ml per 5-ml vial to afford a ready-to-use pharmaceutical nanosuspension with characteristics given in Table 3 and FIGS. 5 and 6.

TABLE-US-00003 TABLE 3 Particle distribution in the pharmaceutical nanosuspension Lot No. Lot No. 740-1-101.1 740-1-101.2 Pharmaceutical nanosuspension (saccharose) (mannitol) pH 7.44 7.29 Particle Particle size, nm 381.7 447.6 distribution PDI 0.255 0.248 (10-l sample) Particle Particle size, nm 386.1 442.6 distribution PDI 0.252 0.252 (30-l sample) HPLC analysis 93.5% 95.0% (percent of the nominal value)

Example 3. Pharmacokinetics of Pharmaceutical Nanosuspensions VM1500LAI and VM1500A-LAI in Dogs

[0045] The nanosuspensions VM1500LAI and VM1500A-LAI obtained in Example 2 were injected to Beagle dogs subcutaneously and intramuscularly as a single dose of 10 mg/kg, and the content of VM1500 in canine plasma was measured for 120 days after the injections. The concentration of tested compounds was measured using the HPLC-MS/MS method.

[0046] To administer the dose, pre-prepared nanosuspensions were injected to the dogs subcutaneously or intramuscularly at 0.1 ml/kg in the same spot. The blood was collected 0, 0.5, 1, 2, 4, 8, 24 h and 2, 3, 6, 8, 12, 15, 18, 22, 29, 36, 43, 50, 57, 64, 71, 78, 85, 92, 107, 114 and 121 days after the injection. The whole blood for plasma production was collected in test tubes containing sodium heparin. Immediately after collecting blood the test tube was cautiously turned over 8 times for better mixing of blood and heparin sodium and then incubated for 15 minutes at room temperature. The plasma was then separated by centrifugation at 3000 rev/min for 10 min. A plasma aliquot of 1 ml was carefully transferred into a cryovial using a self-filling pipette while avoiding contact between the tip and the cell mass/barrier gel. The plasma samples from one Vacuette tube were transferred into two cryovials. The resulting samples were frozen. Prior to analysis, plasma samples and blood cells should be kept at 80 C.

[0047] The concentration of VM-1500 and VM-1500A was measured by means of HPLC-MS/MS. The compound VM-6819 having a similar structure was used as the internal standard (IS). Scanning in a total ion current mode (MS1) allowed us to identify a molecular ion for each tested compound and IS, while basic product ions were recorded in the MS2 mode. To attain maximum sensitivity in the quantitative analysis, the MS/MS procedure was optimized in the MRM mode. For each analyte, at least two MRM transitions were recorded, and one of them was used in quantitative chromatogram processing.

[0048] To achieve the best chromatographic parameters, suitable conditions were chosen for the injection of the test compound and IS in a mixture of MeCN:H.sub.2O (1:1) into the MS/MS detector via an HPLC system. Calibration standards for VM1500 and VM1500A in plasma were prepared in a concentration range of 1.0-2000.0 ng/ml. Along with calibration standards, a zero sample (k0) and k0 with IS were prepared as well as two series of control samples (QC) for low, medium, and high concentrations to serve as the indication of the quality of quantitative calculations. From the stock solutions of VM1500 (2.0 mg/ml) and VM1500A (2.0 mg/ml) in DMSO, 10-fold work dilutions in MeCN:H.sub.2O (1:1) were prepared. Solutions for the calibration standards of (k) and QC samples were prepared from single aliquots of the VM1500 and VM1500A stock solutions.

[0049] Plasma experimental samples used for the preparation of standard samples were thawed at room temperature, stirred and centrifuged for 5 min at 14000 rev/min. Intact plasma (90 l) was transferred into a 1.5-ml test tube, and 10 l of a 10-fold standard dilution of the mixture of test compounds was added to the acetonitrile:water (1:1) mixture. To the experimental sample (90 l), a pure acetonitrile:water (1:1) mixture (10 l) was added. The samples were thoroughly mixed for 10 seconds using a vortex mixer. Then, 10 l of IS (VM-6819 5 mg/ml in acetonitrile:water 1:1) was added, and the mixture was vortexed for another 10 seconds. To the samples, 1 ml of the ethyl acetate:hexane (60:40) mixture was added and mixed in a vortex mixer for 20 sec and then on a shaker at 1000 rev/min for 5 min. The samples were centrifuged at 14000 rev/min for 10 min.

[0050] The organic phase (0.8 ml) was taken to 1.5-ml test tubes and dried under nitrogen flow in an evaporator at 40 C. Dry extracts were diluted in 150 l of the acetonitrile:water (1:1) mixture, vortexed for 10 sec and transferred into a clean plate for an HPLC/MS/MS analysis.

[0051] The concentrations of VM1500 and VM1500A in the samples were estimated from the area-based calibration curve of standard samples chromatographic peaks VM1500 and VM1500A in a matrix (plasma or blood cells) normalized to the IS area. The calibration curve was plotted by fitting a simplest model adequately describing analytical signal versus concentration relationship and using a suitable normalization. Basic pharmacokinetic parameters were found by a model-independent method using the WinNonlin Professional 6.3 software (Pharsight Corporation) based on experimentally derived concentration-time data for each animal: [0052] C.sub.maxmaximum concentration in plasma; [0053] T.sub.maxtime to reach maximum concentration in plasma; [0054] AUC.sub.04area under the PK curve from the moment of drug administration to the last point of concentration measurement; [0055] AUC.sub.0-infarea under the PK curve from the moment of drug administration to infinity; [0056] T.sub.1/2plasma elimination half-life; [0057] k.sub.elelimination rate constant (parameter characterizing the rate of drug elimination from the plasma); [0058] MRTmiddle residence time from the moment of drug administration.

[0059] Statistical processing of the results was performed by means of descriptive statistics. The following parameters were calculated: the arithmetic mean value (M), the standard deviation (SD), the standard error of the mean (SEM), the coefficient of variation (CV), the median. Statistical analysis was carried out using the WinNonlin Professional 6.3 software (Pharsight Corporation).

[0060] Table 4 summarizes pharmacokinetic parameters characterizing the behavior of VM-1500 and VM-1500A after subcutaneous (SC) and intramuscular (IM) injections of the nanosuspensions VM-1500-LAI and VM-1500A-LAI to dogs.

TABLE-US-00004 TABLE 4 Pharmacokinetic parameters VM-1500 and VM-1500A in canine plasma after SC and IM injections of the nanosuspensions VM-1500-LAI and VM-1500A-LAI in a dose of 10 mg/kg SC IM SC IM VM1500- VM1500A- VM1500- VM1500A- VM1500-LAI LAI LAI LAI LAI Parameter Unit VM-1500 VM-1500A AUC.sub.inf h*ng/ml 4 435.7 5 743.6 89 210.0 89 409 121 175.1 83 916 AUC.sub.last h*ng/ml 3 989.9 5 596.7 88 830.4 78 492 120 975.5 79 518 C.sub.max ng/ml 111.4 1 158.7 558.7 104 1 225.0 153 T.sub.1/2 h 229 74.9 170 581 153 446 K.sub.el 1/h 0.003 0.009 0.004 0.001 0.005 0.002 MRT.sub.inf h 180 38.6 205 957 90 657 MRT.sub.last h 95 29.1 200 671 88 537 T.sub.max h 1.7 1.0 64 216 32 64.0

[0061] With intramuscular injection, higher C.sub.max values were observed both for VM-1500 and its metabolite VM-1500A than with subcutaneous injection. For the VM-1500A-LAI formulation, however, the difference was not so appreciable. Furthermore, the total exposure of VM-1500 and VM-1500A in the case of intramuscular injection of VM-1500-LAI was higher than in the case of subcutaneous one, whereas the exposure of VM-1500A was independent of the way of administration of the VM-1500A-LAI formulation. It is to be noted that with intramuscular injection, VM-1500A achieves maximum concentration earlier than with subcutaneous one. Half-life does not substantially depend on the way of administration.

[0062] Average VM-1500A concentrations (C) in canine plasma after single SC and IM injections of the nanosuspensions VM1500-LAI and VM11500A-LAI are given in Table 5 and in FIG. 7.

TABLE-US-00005 TABLE 5 Concentration of VM-1500A in canine plasma after single SC and IM injections of the depot formulations VM-1500-LAI and VM-1500A-LAI at 10 mg/kg VM1500-LAI VM1500-LAI VM1500A-LAI VM1500A-LAI IM SC IM SC Time Mean, SD, Mean, SD, Mean, SD, Mean, SD, hours days ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml ng/ml 0 0.0 0.00 0.00 0.00 0.00 0.00 0.00 1 0.0 55.67 7.76 3.54 0.58 6.35 1.59 3.40 3.48 1 0.0 129.67 12.74 5.62 1.56 11.96 6.64 4.24 2.56 2 0.1 350.67 74.54 16.57 2.75 28.80 9.30 7.12 4.59 4 0.2 482.33 87.93 31.43 6.11 40.23 3.82 12.25 5.03 8 0.3 892.67 108.49 77.93 8.47 84.43 25.20 16.43 3.98 24 1.0 1166.67 151.85 314.67 35.64 124.50 11.46 49.80 13.17 48 2.0 1023.00 187.20 480.00 155.79 131.67 4.25 70.23 16.42 72 3.0 812.67 28.36 535.33 210.24 151.33 18.06 91.73 5.61 144 6.0 245.33 51.60 232.67 8.14 99.37 22.09 59.96 4.82 192 8.0 105.70 26.49 129.00 16.70 115.50 12.26 86.97 3.57 288 12.0 39.43 9.03 90.27 10.83 101.22 8.75 96.22 24.58 360 15.0 12.97 4.16 48.40 7.41 79.38 22.07 57.62 4.52 432 18.0 7.06 2.98 36.10 5.20 61.52 7.52 71.20 4.98 528 22.0 6.04 1.89 34.30 3.70 48.85 7.19 56.42 9.05 696 29.0 2.61 0.84 16.80 4.11 37.07 3.26 65.5 10.8 864 36.0 1.03 0.10 8.57 2.31 27.80 7.89 37.8 3.1 1032 43.0 0.89 0.18 4.83 1.98 32.37 5.09 34.5 12.5 1200 50.0 BLQ 1.47 0.70 13.30 1.31 18.4 3.9 1368 57.0 BLQ BLQ 7.6 2.55 12.02 2.48 1536 64.0 BLQ BLQ 12.27 3.52 12.40 1.60 1704 71.0 BLQ BLQ 8.88 2.74 8.53 1.12 1872 78.0 BLQ BLQ 6.03 1.84 11.24 3.41 2040 85.0 BLQ BLQ 5.87 2.07 9.92 2.52 2208 92.0 BLQ BLQ 6.81 1.46 12.79 3.01 2568 107 BLQ BLQ 6.43 1.38 9.59 0.39 2736 114 BLQ BLQ 9.68 1.33 5.72 1.12 2904 121 BLQ BLQ 5.70 1.81 11.47 1.07 BLQBelow Limit of Quantification

[0063] As can be seen from Table 5 and FIG. 7, after IM injection of the pharmaceutical nanosuspension VM-1500-LAI in a dose of 10 mg/kg to dogs, the effective concentration of the drug VM1500A (IC.sub.50=1.3 nM or 0.74 ng/ml) in plasma is maintained for about a month (after 29 days, C.sub.VM-1500A=2.61 ng/ml), whereas after SC injection, for about a month and a half (after 43 days, C.sub.VM-1500A=4.83 ng/ml).

[0064] An even more convincing result was observed after the IM and SC injections of the nanosuspension VM-1500-LAI in a dose of 10 mg/kg to dogs. In this case, the effective concentration of VM1500A maintained for four months (after 121 days, C.sub.VM-1500A=5.70 ng/ml after IM injection and C.sub.VM-1500A=11.47 ng/ml after SC injection).

INDUSTRIAL APPLICABILITY

[0065] The invention could be used in medicine and veterinary.