PEPTIDE-CONTAINING COMPOSITION FOR USE FOR TREATING NEOPLASTIC LESIONS

Abstract

The present invention provides a pharmaceutical composition comprising the therapeutic peptide CyPep-1, for use in treating neoplastic lesions, in particular warts.

Claims

1. An aqueous pharmaceutical composition, the composition comprising: (i) 0.1-5% therapeutic oligopeptide, wherein the therapeutic oligopeptide comprises the amino acid sequence set forth in SEQ ID NO: 1, or an amino acid sequence having at least 85% sequence identity thereto; (ii) 0.1-5% preservative; (iii) 0.1-5% silicone; (iv) 10-20% di(ethylene glycol) ethyl ether; (v) 0.005-0.1% butylated hydroxyanisole; (vi) 1-10% liquid paraffin; (vii) 1-10% soft paraffin; (viii) 1-10% cetostearyl alcohol; and (ix) 0.1-5% polyoxyl 20 cetostearyl ether.

2. The composition of claim 1, comprising: (i) 0.5-2% therapeutic oligopeptide; (ii) 0.5-2% preservative; (iii) 0.5-2% silicone; (iv) 14-16% di(ethylene glycol) ethyl ether; (v) 0.01-0.05% butylated hydroxyanisole; (vi) 4-6% liquid paraffin; (vii) 5-7% soft paraffin; (viii) 4.5-6.5% cetostearyl alcohol; and (ix) 1-3% polyoxyl 20 cetostearyl ether.

3. The composition of claim 1 or 2, wherein the therapeutic oligopeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1.

4. The composition of any one of claims 1 to 3, wherein the oligopeptidic compound is an inverso-compound, every amino acid of which is a D-amino acid.

5. The composition of any one of claims 1 to 4, wherein the preservative is phenoxyethanol.

6. The composition of any one of claims 1 to 5, wherein the silicone is a dimethicone.

7. The composition of claim 6, wherein the dimethicone is dimethicone with a viscosity of 350 cSt at 25° C. (dimethicone 350 cSt).

8. The composition of any one of claims 1 to 7, wherein the cetostearyl alcohol is a 50:50 mixture of cetyl alcohol and stearyl alcohol (cetostearyl alcohol 50:50).

9. The composition of any one of claims 1 to 8, wherein the soft paraffin is white soft paraffin.

10. The composition of any one of claims 1 to 9, wherein the composition consists of: (i) 1% therapeutic oligopeptide, wherein the therapeutic oligopeptide consists of the amino acid sequence set forth in SEQ ID NO: 1 and is an inverso-compound, every amino acid of which is a D-amino acid; (ii) 1% phenoxyethanol; (iii) 1% dimethicone 350 cSt; (iv) 15% di(ethylene glycol) ethyl ether; (v) 0.02% butylated hydroxyanisole; (vi) 4.8% liquid paraffin; (vii) 6.3% white soft paraffin; (viii) 5.5% cetostearyl alcohol 50:50; (ix) 2.4% polyoxyl 20 cetostearyl ether; and (x) 62.98% water.

11. A composition as defined in any one of claims 1 to 10 for use in therapy.

12. A composition as defined in any one of claims 1 to 10 for use in the treatment of a neoplasm.

13. The composition for use according to claim 12, wherein the neoplasm is a wart.

14. The composition for use according to claim 13, wherein the treatment comprises topical administration of the composition to the wart.

15. The composition for use according to claim 14, wherein the treatment comprises daily administration of the composition to the wart for a period of about 4 weeks.

16. The composition for use according to any one of claims 13 to 15, wherein the wart is a cutaneous wart.

17. The composition for use according to claim 12, wherein the neoplasm is a neoplasm of the skin.

18. The composition for use according to claim 17, wherein the neoplasm of the skin is an actinic keratosis, a seborrheic keratosis, Bowens disease, basal cell carcinoma or squamous cell carcinoma.

19. A method of treating a neoplasm in a subject, comprising administering a composition as defined in claims 1 to 10 to the subject.

20. Use of a composition as defined in any one of claims 1 to 10 in the manufacture of a medicament for treating a neoplasm.

21. The method of claim 19 or the use of claim 20, wherein the neoplasm is a wart.

22. The method or use of claim 21, wherein treatment of the wart is as defined in claim 14 or 15, and/or the wart is as defined in claim 16.

23. The method of claim 19 or the use of claim 20, wherein the neoplasm is as defined in claim 17 or 18.

Description

FIGURE LEGENDS

[0138] FIG. 1 shows the results of calibration experiments to determine CyPep-1 concentration by HPLC (using the method of Example 1). A shows a representative calibration curve of CyPep-1 standards prepared in deionised water over a concentration range of 2.5-500 pg/ml. The points shown in the graph are the mean values of 3 HPLC runs. B shows a sample chromatogram representative of a CyPep-1 calibration standard (in this case with a concentration of 100 pg/ml CyPep-1).

[0139] FIG. 2 shows the mean cumulative amount of CyPep-1 released per unit area (μg/cm.sup.2) between 1 and 7 hours (presented as the square root of time) from 5 tested pharmaceutical formulations across an isopore membrane into a receptor solution (deionised water). Data presented as mean±standard deviation (n=6).

EXAMPLES

Example 1

Development of Analytical Method

[0140] To measure stability of CyPep-1 in the tested pharmaceutical formulations, the following analytical method for quantification of CyPep-1 by HPLC was developed. Various conditions (temperature, flow rate etc.) were tested (data not shown) and the following procedure was found to be optimal.

[0141] HPLC was performed using a Phenomenex (USA) Kinetex C18 5 μm 100 Å 4.6×250 mm column and a Phenomenex SecurityGuard C18 5 μm guard column, as follows:

TABLE-US-00001 Mobile phase A 0.05% v/v trifluoroacetic acid in water Mobile phase B 0.05% v/v trifluoroacetic acid in acetonitrile Initial flow rate 1.0 mL/min Run time 30 min Wavelength 276 nm Column temperature 25° C. Auto sampler temperature  5° C. % % Time Mobile Mobile (min) Phase A Phase B Flow gradient 0.00 90 10 3.00 90 10 24.00 70 30 26.00 70 30 26.01 90 10 30.00 90 10 Injection volume 25 μL Retention time of CyPep-1 ~19.4 minutes Sample and standard diluent Deionised water Seal wash and line storage 20:80 v/v methanol:water

[0142] This HPLC procedure was determined to have a lower limit of quantification of CyPep-1 of 2.5 μg/ml. A representative calibration curve of CyPep-1 standards and a sample chromatogram representative of a calibration standard obtained using the above-described HPLC method are shown in FIG. 1A and 1B, respectively.

Example 2

Formulation Development

[0143] Initial experiments demonstrated high solubility of CyPep-1 in deionised water (pH 4, 5, 6 or 7), ethanol, phenoxyethanol and benzyl alcohol (saturated solubility of CyPep-1 in each of more than 10% w/w). The stability of CyPep-1 in these liquids was tested. Good recovery (at least 95%) of CyPep-1 was observed following 4 weeks storage at 40° C. and 50° C. in deionised water (pH 4-7) and at 40° C. in a mixture of ethanol and phenoxyethanol.

Solubility Testing—Method

[0144] The saturated solubility of CyPep-1 in various liquid excipients was assessed as follows: [0145] (i) CyPep-1 (ca. 20.0 mg) was weighed into suitably sized glass vials. [0146] (ii) Each of the excipients (ca. 500 mg) were added to the individual glass vials from Step (i). [0147] (iii) The CyPep-1 and excipient systems were stirred for 24 h (once saturation was observed) in a pre-calibrated water bath at 25° C. [0148] (iv) During the 24 h of stirring the solutions were visually inspected hourly (where possible) to observe if the CyPep-1 had dissolved in the excipients. [0149] (v) If CyPep-1 was observed to dissolve then ca. 20.0 mg increments of CyPep-1 were added to the vial until saturation was reached or until a total of 60 mg had been added at which point the solubility was reported as % w/w. [0150] (vi) For saturated systems any un-dissolved CyPep-1 was removed from the saturated solution via centrifugation for 10 min at ca. 16,000×g. A sample of the saturated supernatant was examined using light microscopy with a magnification of 200-1000× using a Leica (Germany) DME light microscope. The saturated supernatant was mounted on a microscope slide and examined immediately using both polarised and non-polarised light for the presence of CyPep-1 crystals. The dilution (Step (vii)) was performed immediately after the centrifugation step. [0151] (vii) Since the solubility of CyPep-1 in these excipients was unknown, an appropriate dilution was made to achieve a concentration of CyPep-1 in sample diluent (deionised water) that was above the limit of quantification of the HPLC method, prior to analysis. For excipients which were saturated following the addition of ca. 20 mg of CyPep-1 the following dilution was performed: [0152] (a) Into a 10 mL volumetric flask ca. 50 mg of the saturated solution was weighed. [0153] (b) The volumetric flask from Step (a) was made to volume with sample diluent (deionised water) and vortex mixed for 30 s. [0154] (c) An aliquot of the diluted sample from Step (b) was transferred to an HPLC vial and analysed by HPLC as described above. [0155] (viii) For excipients which were saturated following the addition of ca. 40 mg of CyPep-1 the dilution was performed as detailed in Step (vii), however a 20 mL volumetric flask was used.

Stability Testing—Method

[0156] The stability of CyPep-1 was assessed in the liquid excipients which demonstrated a saturated solubility of CyPep-1 above 10% (w/w). The stability of CyPep-1 in the excipients was assessed as follows: [0157] (i) CyPep-1 (40.0±0.1 mg) was weighed into a suitably sized glass vial. [0158] (ii) Each of the excipients (39.96±0.2 g) were added to each of the suitably sized glass vials from Step (i). [0159] (iii) A PTFE magnetic stirrer was added to the mixtures of Step (ii), and left to mix for ca. 16 h. [0160] (iv) The solutions from Step (iii) were transferred to 3 mL borosilicate glass vials with Teflon-lined lids. Sufficient vials were prepared for testing at t=0 and 2 additional time points at 25, 40 and 50° C.; n=1 aliquot per time point was prepared. [0161] (v) Placebo solutions (no added CyPep-1) were also prepared and placed onto stability according to Step (iv). [0162] (vi) The time points assessed were t=0, 2 and 4 weeks, following storage at 40 and 50° C. At each time point the CyPep-1 content and % a/a in the solutions was analysed as follows: [0163] a) Each CyPep-1 solution (500±10 mg) was weighed into three individual 10 mL volumetric flasks. [0164] b) The volumetric flasks were made to volume with sample diluent (deionised water) and thoroughly mixed by vortex mixing for 30 s followed by inversion 5 times. [0165] c) If the excipient was observed to be immiscible with the sample diluent (Step (b)), the extraction was stirred at ambient temperature for ca. 2 h, prior to centrifugation for 10 min at ca. 16,000×g. [0166] d) An aliquot of each sample (from Step (b) or (c)) was transferred to amber HPLC vials and analysed using HPLC as described in Example 1. [0167] e) For the placebo solutions Steps (a) to (d) were followed, however, only one replicate was prepared and analysed.

[0168] Based on these initial solubility and stability experiments, several CyPep-1 formulations were synthesised for further testing, as follows:

Cream 01:

[0169]

TABLE-US-00002 Concentration Ingredient (% w/w) CyPep-1 1 Deionised Water 24 PEG 400 42 Transcutol-P 12 Benzyl Alcohol 1 Cetostearyl Alcohol 5 Crodamol GTCC 10 Dimethicone 1 Span-60 1.4 Tween-60 2.6

Cream 02:

[0170]

TABLE-US-00003 Concentration Ingredient (% w/w) CyPep-1 1 Deionised Water 24 PEG 400 42 Transcutol-P 12 Benzyl Alcohol 1 Cetostearyl Alcohol 5 Crodamol GTCC 10 Dimethicone 1 Brij-S2 1.5 Brij-S20 2.5

Cream 03:

[0171]

TABLE-US-00004 Concentration Ingredient (% w/w) CyPep-1 1 Deionised Water 24 PEG 400 42 Transcutol-P 12 Benzyl Alcohol 1 Cetostearyl Alcohol 5.5 Liquid Paraffin 4.8 White Soft Paraffin 6.3 Dimethicone 1 Ceteareth-20 2.4

Cream 04:

[0172]

TABLE-US-00005 Concentration Ingredient (% w/w) CyPep-1 1 Deionised Water 30 PEG 400 32.98 Transcutol-P 15 Phenoxyethanol 1 Cetostearyl Alcohol 5 BHA 0.02 Crodamol GTCC 10 Dimethicone 1 Brij-S2 1.5 Brij-S20 2.5

Cream 05:

[0173]

TABLE-US-00006 Concentration Ingredient (% w/w) CyPep-1 1 Deionised Water 78 Phenoxyethanol 1 Liquid Paraffin 11.17 Cetostearyl Alcohol 3.83 Dimethicone 1 Brij-S2 1.4 Brij-S20 2.6

Cream 06:

[0174]

TABLE-US-00007 Ingredient Concentration (% w/w) CyPep-1 1 Deionised Water 78 Phenoxyethanol 1 Cetostearyl Alcohol 5 Crodamol GTCC 10 Dimethicone 1 Brij-S2 1.5 Brij-S20 2.5

Cream 07:

[0175]

TABLE-US-00008 Ingredient Concentration (% w/w) CyPep-1 1 Deionised Water 77 Benzyl Alcohol 2 Cetostearyl Alcohol 5 Crodamol GTCC 10 Dimethicone 1 Brij-S2 1.5 Brij-S20 2.5

Cream 08:

[0176]

TABLE-US-00009 Ingredient Concentration (% w/w) CyPep-1 1 Deionised Water 78 Phenoxyethanol 1 Cetostearyl Alcohol 5.5 Liquid Paraffin 4.8 White Soft Paraffin 6.3 Ceteareth-20 2.4 Dimethicone 1

Cream 09:

[0177]

TABLE-US-00010 Ingredient Concentration (% w/w) CyPep-1 1 Deionised Water 62.98 Phenoxyethanol 1 Transcutol-P 15 Cetostearyl Alcohol 5.5 Liquid Paraffin 4.8 White Soft Paraffin 6.3 BHA 0.02 Ceteareth-20 2.4 Dimethicone 1

Cream 10:

[0178]

TABLE-US-00011 Ingredient Concentration (% w/w) CyPep-1 1 Deionised Water 41.98 PEG 400 35 Benzyl Alcohol 2 BHA 0.02 Cetostearyl Alcohol 5.5 Liquid Paraffin 4.8 White Soft Paraffin 6.3 Ceteareth-20 2.4 Dimethicone 1

Ointment 1:

[0179]

TABLE-US-00012 Ingredient Concentration (% w/w) CyPep-1 1 PEG 400 58 Transcutol-P 15 Benzyl Alcohol 1 PEG 3350 25

Ointment 2:

[0180]

TABLE-US-00013 Ingredient Concentration (% w/w) CyPep-1 1 PEG 400 49 Transcutol-P 15 Deionised Water 10 PEG 4000 25

Ointment 3:

[0181]

TABLE-US-00014 Ingredient Concentration (% w/w) CyPep-1 1 PEG 400 62.98 Phenoxyethanol 1 BHA 0.02 Deionised Water 10 PEG 4000 25

Aqueous Gel 1:

[0182]

TABLE-US-00015 Ingredient Concentration (% w/w) CyPep-1 1 Deionised Water 30 PEG 400 52 Transcutol-P 15 Benzyl Alcohol 1 Carbopol 974 1

Aqueous Gel 2:

[0183]

TABLE-US-00016 Ingredient Concentration (% w/w) CyPep-1 1 Deionised Water 96 Benzyl Alcohol 2 Carbopol 974 1

Aqueous Gel 3:

[0184]

TABLE-US-00017 Ingredient Concentration (% w/w) CyPep-1 1 Deionised Water 96 Benzyl Alcohol 2 Hydroxyethyl Cellulose 1

Aqueous Gel 4:

[0185]

TABLE-US-00018 Ingredient Concentration (% w/w) CyPep-1 1 Deionised Water 85.98 BHA 0.02 Ethanol 10 Phenoxyethanol 1 Hydroxyethyl Cellulose 2

Aqueous Gel 5:

[0186]

TABLE-US-00019 Ingredient Concentration (% w/w) CyPep-1 1 Deionised Water 70.98 BHA 0.02 Ethanol 10 Transcutol-P 15 Phenoxyethanol 1 Hydroxyethyl Cellulose 2

Non-Aqueous Gel 1:

[0187]

TABLE-US-00020 Ingredient Concentration (% w/w) CyPep-1 1 PEG 400 68.1 Ethanol 4.9 Transcutol-P 25 Hydroxypropyl Cellulose 1

Example 3

Formulation Testing

[0188] Ointment 1 and Non-Aqueous Gel 1 were immediately discarded, since CyPep-1 proved insoluble in these formulations. Formulations were then centrifuged at 16 minutes. Formulations which separated during that time were deemed unstable and discarded. The following formulations were discarded as deemed unstable due to separation during centrifugation: Cream 01 and Ointment 02. Aqueous Gels 01 and 02 were discarded due to turbidity, indicating the gelling agent (Carbopol 974) was not fully hydrated. It was determined that this was due to incompatibility of Carbopol 974 with CyPep-1. Creams 2 and 3, and Aqueous Gel 3, were also discarded. Stability of CyPep-1 in the various formulations was then investigated.

[0189] CyPep-1 Extraction—Method

[0190] Prior to stability testing, a CyPep-1 extraction method was generated. Several methods were tested (data not shown). The optimal extraction method developed was performed as follows: [0191] (i) 150 mg formulation was weighed into a 10 ml volumetric flask. [0192] (ii) The flask was made up to volume with 50:50 v/v water:ethanol. [0193] (iii) The volumetric flask was vortex-mixed for 30 s to disperse the formulation, yielding an extraction solution containing CyPep-1 at a concentration of 150 μg/ml. [0194] (iv) A PTFE magnetic stirrer was added to the volumetric flask from Step (iii) and the contents of the volumetric flask was stirred for 2 h at 500 rpm. [0195] (v) The extraction solution from Step (iv) was transferred to centrifuge tubes and the samples were centrifuged at 16,000×g for 10 min at 25° C. [0196] (vi) The supernatant was filtered using a 0.2 μm PTFE filter.

[0197] The obtained, filtered solution was used for HPLC analysis. This method was found to recover an estimated 96% of CyPep-1 from the formulations.

[0198] Short-Term Stability Testing

[0199] The 10 formulations described above which were not discarded were taken into stability testing. Samples of the formulations were stored at 2-8° C., 25° C. and 40° C. After 2 weeks, samples stored at 25° C. and 40° C. were assessed; after 4 weeks and 11 weeks samples stored at 2-8° C. and 25° C. were assessed.

[0200] No change in macroscopic (by eye) or microscopic (400× magnification) appearance was seen for any formulation at any time point.

TABLE-US-00021 Percentage recovery of CyPep-1 (as a percentage of the theoretical concentration) in formulations at t = 0 and following storage for t = 2 weeks at 25 and 40° C. and t = 4 and 11 weeks at 2-8 and 25° C. T = 2 weeks T = 4 weeks T = 11 weeks Formulation T = 0* 25° C. 40° C. 2-8° C. 25° C. 2-8° C. 25° C. Cream 04 103.15 95.89 66.45 98.16 96.72 101.24 106.38 (101.95-105.45) (95.43-96.35) (58.50-74.40) (97.89-98.43) (96.46-96.98) (101.18-101.30) (105.77-107.00) Cream 05 100.54 96.30 77.37 93.82 97.29 93.48 98.41 (99.37-101.85) (95.97-96.62 (76.33-78.42) (93.54-94.11) (96.41-98.18) (87.92-99.05) (97.29-99.54) Cream 06 101.27 94.39 70.41 97.56 88.01 97.15 95.89 (98.11-105.51) (93.93-94.84 (66.76-74.05) (96.59-98.54) (80.76-95.25) (97.14-97.16) (95.53-96.25) Cream 07 101.27 91.80 73.08 95.27 94.64 95.97 92.02 (99.33-103.31) (91.71-91.89) (68.98-77.19) (94.72-95.82) (94.19-95.09) (95.18-96.76) (91.80-92.24) Cream 08 100.58 97.77 73.85 96.10 97.57 99.56 96.80 (98.52-103.25) (97.57-97.98 (72.44-75.25) (93.98-98.21) (96.15-99.00) (98.97-100.15) (96.41-97.19) Cream 09 100.37 99.71 76.27 96.16 98.77 100.96 97.06 (97.43-104.87) (99.38-100.04) (74.59-77.96) (93.50-98.82 (98.41-99.13) (99.81-102.11) (96.46-97.67) Cream 10 100.19 98.69 78.11 101.67 101.18 102.02 101.08 (98.60-102.63) (98.48-98.90 (76.55-79.68) (100.92-102.42) (100.93-101.43) (101.93-102.12) (100.77-101.39) Ointment 3 96.21 91.94 67.83 97.27 83.57 100.69 82.50 (96.21-96.21) (91.29-92.58) (66.35-69.30) (96.76-97.77) (81.05-86.09) (99.16-102.23) (82.29-82.71) Aqueous 94.14 102.01 100.91 100.18 98.75 97.24 105.36 Gel 4 (91.68-96.59) (99.67-104.35) (99.10-102.71) (100.03-100.34) (98.33-99.18) (95.34-99.14) (105.09-105.62) Aqueous 74.26 99.16 97.34 101.58 96.68 99.47 106.26 Gel 5 (71.04-77.10) (98.09-100.24) (94.75-99.92) (99.97-103.19) (96.03-97.34) (99.42-99.52) (106.18-106.35) *n = 3

[0201] At t=0 the percentage recovery of CyPep-1 for all of the formulations prepared for short term stability was observed to be 94.14-103.15% of the theoretical concentration. The exception to this was formulation Aqueous Gel 5, which resulted in a recovery of 74.26% of the theoretical concentration of CyPep-1 at t=0. However the results for subsequent time points were between 96.68 and 106.26% of the theoretical concentration of CyPep-1, therefore the result obtained at t=0 was likely to be an anomalous result.

[0202] Following storage for t=2 weeks at 40° C. a drop in the recovery of CyPep-1 was observed for all of the cream and ointment formulations assessed (66.45-78.11%). Based on these results, the remainder of the stability assessments were performed at 2-8 and 25° C. as CyPep-1 was deemed to be unstable at 40° C. Following storage for t=2, 4 and 11 weeks at 25° C. and t=4 and 11 weeks at 2-8° C. no substantial changes (<10%) were observed in the percentage recovery of CyPep-1, demonstrating that CyPep-1 was stable in these formulations for up to 11 weeks under the storage conditions tested. The exception to this was Ointment 3 which demonstrated a downwards trend in recovery of CyPep-1 over time when stored at 25° C.

[0203] Based on a combination of these results, and the aesthetics of each formulation, the following formulations were selected for further study:

[0204] Cream 05

[0205] Cream 09

[0206] Cream 10

[0207] Aqueous Gel 4

[0208] Aqueous Gel 5.

[0209] The following formulations were discarded:

[0210] Cream 04: this demonstrated relatively poor stability, and the formulation was found to be aesthetically poor (off-white and greasy).

[0211] Cream 06: this demonstrated relatively poor stability.

[0212] Cream 07: this demonstrated relatively poor stability.

[0213] Cream 08: although this showed equal stability to the selected formulations, the formulation does not contain an antioxidant. Oxidation was found to be a potential route of CyPep-1 oxidation (data not shown) and so this formulation was discarded.

[0214] Ointment 3: this demonstrated relatively poor stability, and the formulation was found to be aesthetically poor (off-white and greasy).

Example 4

In Vitro Drug Release Testing

[0215] CyPep-1 was found to display minimal non-specific binding to glass and plastic, meaning no detergent was required in the receptor solution to prevent non-specific binding. Deionised water was therefore selected as the receptor solution for the study.

[0216] Five synthetic membranes were then evaluated for back-diffusion of the formulations. The membranes tested were supor, nitrocellulose, isopore, cellulose and nylon. Only the cellulose membrane demonstrated back diffusion following application of the formulation with deionised water as a receptor solution, potentially altering the composition of the formulation applied. No back diffusion was noted with any other membrane tested.

[0217] The same membranes were evaluated for binding of CyPep-1. Recovery of CyPep-1 following incubation with the membranes is shown below (mean recovery, n=3, compared to control with no membrane, incubation at 32° C.):

TABLE-US-00022 Membrane 24 Hours 48 Hours 96 Hours Supor 81.53 70.39 86.13 Nitrocellulose 74.67 65.62 76.03 Isopore 93.61 89.52 88.61 Cellulose 38.32 18.54 18.6 Nylon 82.77 59.6 77.54

[0218] The isopore membrane was selected for use in the drug release study, since CyPep-1 recovery was highest from this membrane, and no back-diffusion was seen with this membrane.

In Vitro Drug Release Experiment—Method

[0219] Individually calibrated static Franz cells were employed in the method, each cell having an average surface area and volume of about 2 cm.sup.2 and 10 ml, respectively. The static Franz cells were mounted with isopore membrane and filled with deionised water. The static Franz cells were then equilibrated to 32° C. in a water bath, and 30 minutes later dosed with an “infinite dose” (300 mg/cm.sup.2) of a test formulation. One Franz cell was mounted with isopore membrane but not dosed with a formulation, in order to act as a blank.

[0220] At each time point (0, 1, 2, 3, 4, 5, 6 and 7 hours) a 1 ml aliquot of receiver fluid (deionised water) was removed from the Franz cell using a syringe, via the sampling arm. Each 1 ml aliquot was replaced with 1 ml pre-warmed deionised water, to retain a constant volume, ensuring no bubbles were generated in the cell. Each aliquot removed was analysed by HPLC, using the method described above, and the cumulative amount of CyPep-1 released across the isopore membrane calculated. For the duration of the experiment, the Franz cells were protected from light by occlusion with parafilm.

[0221] In Vitro Drug Release Experiment—Results

[0222] The amount of CyPep-1 released across the isopore membrane into the receptor solution over the course of the 7 hour experimental period by each formulation is shown in FIG. 2. As shown, The gel formulations Aqueous Gel 4 and Aqueous Gel 5 were observed to release the highest level of CyPep-1 of the tested formulations. These formulations were observed to have a rapid release of CyPep-1 over the intial 1 h experimental period, followed by minimal additional release thereafter. The cream formulations Cream 05, Cream 09 and Cream 10 initially released a lower amount of CyPep-1 at the 1 h time point when compared to the gel formulations, but continued to release CyPep-1 between the 1 and 7 h time points.

[0223] These experiments showed that Cream 09 clearly had the most advantageous properties for use as a pharmaceutical. As shown in Example 3, above, CyPep-1 is very stable in this cream. The results shown in Example 4 demonstrate a high level of sustained release of CyPep-1 from the cream. Sustained release of CyPep-1 is desirable, since it means that following a single application of the formulation to a wart CyPep-1 is continuously being released onto the wart, meaning that each application of cream has a long-lasting therapeutic effect. This in turn means that a lower frequency of application of the cream is required than for a formulation such as the tested aqueous gels which release their CyPep-1 content essentially instantaneously, and therefore have only a relatively brief therapeutic effect on the treated wart. Cream 09 releases comfortably the highest level of CyPep-1 of the tested creams (each of which demonstrates sustained release of the peptide), and therefore has the most advantageous properties of all tested formulations, and can be expected to display the highest level of efficacy in wart treatment.

Example 5

Manufacture of Cream 09

[0224] Two vessels were utilised during the manufacturing process, Vessel A and Vessel B. Phenoxyethanol and Transcutol-P were added to Vessel A, followed by BHA. The contents of the vessel were stirred until BHA was solubilised. Water was then added to Vessel A, followed by CyPep-1. The mixture was stirred until a clear solution was obtained (free from particles or crystals).

[0225] The liquid paraffin, soft white paraffin, cetostearyl alcohol and polyoxyl 20 cetostearyl ether were added to Vessel B. The contents of Vessel B were heated to 65° C., and melted. The contents of Vessel A were also heated to 65° C. The contents of Vessel B were then added to Vessel A and homogenised for mins at 10,000 rpm. During the homogenisation process dimethicone was added. The formulation was then cooled to room temperature, while stirring continuously.

Example 6

Wart Treatment with Cream 09

[0226] A clinical trial was carried out testing the effect of CyPep-1, in the context of Cream 09, on cutaneous warts. Patients were split into two groups: one group received Cream 09, the other a placebo (Placebo 09, equivalent to Cream 09 except CyPep-1 is replaced with water).

[0227] The creams were administered to the patients once daily, by direct topical administration to the treated wart(s), for a duration of 28 days. Following application of the cream, the cream-covered wart was covered with a transparent film for 8-12 hours, to prevent loss of the cream from the wart. Patients were followed up weekly.