NL2024834B1 - Pharmaceutical excipients - Google Patents

Abstract

The invention pertains to a pharmaceutical adjuvant composition comprising an emulsion of a lipid composition, in the form of an oil derived from insect tissue, in water; and a pharmaceutical adjuvant, in the emulsion. The invention also pertains to a pharmaceutical composition comprising an emulsion of a lipid composition, in the form of an oil derived from insect tissue, and a therapeutic or prophylactic agent, and to the use of a lipid composition in the form of an oil derived from insect tissue in the manufacture of a pharmaceutical adjuvant composition for enhancing or promoting an immune response in a subject, wherein the immune response is stimulated by administration of a vaccine, immunomodulator or other therapeutic agent to the subject. In one embodiment the insect tissue from which the lipid composition is derived is raw larvae of the black soldier fly (Hermetia illucens).

Description

NL31175-HS

PHARMACEUTICAL EXCIPIENTS

FIELD OF THE INVENTION THIS INVENTION relates to pharmaceutical excipients. The invention provides a pharmaceutical excipient and also provides for use thereof. The invention further provides a pharmaceutical composition and also provides for use thereof. The invention further provides a medicament and also provides for use thereof. Further, the invention provides a method of treatment of the human or animal body by therapy or prophylaxis.

BACKGROUND TO THE INVENTION ADJUVANTS HAVE BECOME KEY COMPONENTS of vaccines for the enhancement of immune responses. This is at least since the inherent immune potentiation of live or killed bacterial or viral vaccines has been lost with the introduction of more advanced and safer recombinant vaccine technology. Adjuvants have been proven to enhance the magnitude, breadth, quality and longevity of specific immune responses to antigens produced using such technologies.

Adjuvants can be formulated in numerous ways, including as an emulsion. The use of biodegradable shark squalene, derived from shark liver, as an oil excipient in such emulsions has become popular, since it has a desirably small reactogenic profile when compared to mineral and vegetable oils.

However, the environmental and economical sustainability of the use of shark squalene needs to be carefully considered. In this regard, it has been shown that squalene can be successfully derived from olives. There is, however, a need for alternative excipients and adjuvants.

SUMMARY OF THE INVENTION IN ACCORDANCE WITH A FIRST ASPECT OF THE INVENTION IS PROVIDED a pharmaceutical excipient comprising a lipid composition derived from insect tissue. The lipid composition may comprise a composition of fatty acids. The fatty acids may originate from the insect tissue. In the term “composition of fatty acids’ the word ‘of’ does not strictly mean, although its meaning includes, that the composition of fatty acids consists exclusively of fatty acids. Other compounds may also be included. Typically, the composition of fatty acids would consist mainly of fatty acids, i.e. greater than 50% by weight, e.g. 90% by weight. The lipid composition may be in the form of an oil. In some embodiments of the invention, the oil may be a solid fat at room temperature. The insect tissue may be raw insect tissue. In one embodiment of the invention, the insect tissue may be insect larvae. For example, the insect larvae may be larvae of the black soldier fly (Hermetia illucens).

Being derived from insect tissue, the lipid composition may have been produced by processing insect tissue, more specifically by processing raw insect tissue.

Processing the insect tissue to produce the lipid composition may, for example, have included pressing the insect tissue, thus obtaining the lipid composition. Typically, such pressing may have produced the lipid composition as an oil, separate of the insect tissue. Thus, the oil would not typically include insect tissue.

Processing may also have included purifying the oil after pressing, e.g. by cooking, filtering, centrifuging, or a combination thereof. Thus, deriving the lipid composition from insect tissue may, for example, have comprised pressing the insect tissue, thus obtaining a liquid product and residual insect tissue; separating the liquid product from residual insect tissue; and recovering the lipid composition, in the form of an oil, from the liquid product. The pharmaceutical excipient may, in one embodiment of the invention, be formulated as an emulsion. For example, the pharmaceutical excipient may be formulated as a water-in-oil emulsion, as an oil-in-water emulsion, or as a water-in-oil-in-water emulsion. The oil phase would, typically, comprise the lipid composition.

The pharmaceutical excipient may, in one embodiment of the invention, comprise a pharmaceutical adjuvant. Thus, the pharmaceutical excipient may be a pharmaceutical adjuvant composition. THE INVENTION THEREFORE EXTENDS, AS A SECOND ASPECT THEREOF, TO a pharmaceutical adjuvant composition comprising a pharmaceutical adjuvant; and a pharmaceutical excipient according to the first aspect of the invention.

In the term “pharmaceutical adjuvant’ the word “pharmaceutical” is used merely to specify the field of application of the adjuvant, which is the pharmaceutical field. This distinguishes from adjuvants that find application in other fields, for example in the field of pesticides.

Broadly, in the context of the present invention, a pharmaceutical adjuvant is understood to be a pharmacological or immunological agent that modifies the effect of other such agents.

The adjuvant may, in particular, be an immunologic adjuvant.

Thus, the pharmaceutical adjuvant composition may, in one embodiment thereof, comprise an emulsion of a lipid composition, in the form of an oil derived from insect tissue, in water; and a pharmaceutical adjuvant, in the emulsion.

The pharmaceutical adjuvant composition may, itself, serve as a pharmaceutical excipient, particularly when co-formulated with a therapeutic or prophylactic agent as a pharmaceutical composition, e.g. as hereinafter described.

5 IN ACCORDANCE WITH A THIRD ASPECT OF THE INVENTION, THERE IS PROVIDED a pharmaceutical composition comprising a pharmaceutical excipient according to the first aspect of the invention, optionally in the form of a pharmaceutical adjuvant composition according to the second aspect of the invention; and a therapeutic or prophylactic agent.

The therapeutic or prophylactic agent may, for example, be an agent that provides acquired immunity to a human or animal when administered to the human or animal, such as an inactivated or attenuated compound from a micro-organism, an antigen, an immunomodulator, or the like. Alternatively, or in addition, the therapeutic or prophylactic agent may be an active pharmaceutical ingredient.

Broadly, in the context of the present invention, an immunomodulator is understood to be a substance that modulates or influences the immune system by stimulating an immune response in a subject.

In one embodiment of the invention, the therapeutic or prophylactic agent may be a vaccine. In such a case, the pharmaceutical excipient may be in the form of a pharmaceutical adjuvant composition, as described in accordance with the second aspect of the invention. The pharmaceutical adjuvant may then be one that enhances or increases the effectiveness of the vaccine. The vaccine would preferably be one with which the pharmaceutical excipient has, but for the adjuvant, a decreased or subdued action, such that the pharmaceutical activity of the vaccine is not materially affected by the pharmaceutical excipient, but for the adjuvant.

The pharmaceutical composition may be formulated for simultaneous or sequential administration of the pharmaceutical excipient, optionally as the pharmaceutical adjuvant composition, and the therapeutic or prophylactic agent, jointly or severally.

IN ACCORDANCE WITH A FOURTH ASPECT OF THE INVENTION THERE IS PROVIDED a pharmaceutical adjuvant composition according to the second aspect of the invention for use in a method of enhancing or promoting an immune response in a subject, wherein the immune response is stimulated by administration of a vaccine, an immunomodulator, or other therapeutic agent to the subject.

IN ACCORDANCE WITH A FIFTH ASPECT OF THE INVENTION THERE IS PROVIDED use of the lipid composition according to the first aspect of the invention in the manufacture of a pharmaceutical adjuvant composition for enhancing or promoting an immune response in a subject, wherein the immune response is stimulated by administration of a vaccine, an immunomodulator, or other therapeutic agent to the subject.

IN ACCORDANCE WITH A SIXTH ASPECT OF THE INVENTION THERE IS PROVIDED a pharmaceutical adjuvant composition according to the second aspect of the invention or a pharmaceutical composition according to the third aspect of the invention, for use in a method of treatment against disease, practiced on the human or animal body by therapy or prophylaxis.

IN ACCORDANCE WITH A SEVENTH ASPECT OF THE INVENTION, THERE IS PROVIDED use of a lipid composition derived from insect tissue in the manufacture of a pharmaceutical adjuvant composition for treating the human or animal body against disease, by therapy or prophylaxis.

The pharmaceutical adjuvant composition may be in accordance with the second aspect of the invention. IN ACCORDANCE WITH AN EIGTH ASPECT OF THE INVENTION, THERE IS PROVIDED use of a lipid composition derived from insect tissue; or a pharmaceutical excipient according to the first aspect of the invention; or a pharmaceutical adjuvant composition according to the second aspect of the invention in the manufacture of a pharmaceutical composition for treating the human or animal body against disease, by therapy or prophylaxis. The lipid composition may be as hereinbefore described. The pharmaceutical adjuvant composition may be a pharmaceutical adjuvant composition according to the second aspect of the invention.

The pharmaceutical composition may be a pharmaceutical composition according to the third aspect of the invention.

IN ACCORDANCE WITH A NINTH ASPECT OF THE INVENTION THERE IS PROVIDED a method of treatment practiced on the human or animal body, by therapy or prophylaxis, the method including administering to a human or animal a pharmaceutical adjuvant composition according to the second aspect of the invention; or a pharmaceutical composition according to the third aspect of the invention.

IN ACCORDANCE WITH A TENTH ASPECT OF THE INVENTION THERE IS PROVIDED use of a pharmaceutical adjuvant composition according to the second aspect of the invention; or a pharmaceutical composition according to the third aspect of the invention in the treatment of disease in on the human or animal body, by therapy or prophylaxis.

IN ACCORDANCE WITH AN ELEVENTH ASPECT OF THE INVENTION THERE IS PROVIDED a method of producing a pharmaceutical excipient, the method including emulsifying water and a lipid composition, in the form of an oil, derived from insect tissue, to produce an emulsion of the water and the lipid composition. The lipid composition may be as hereinbefore described.

The emulsion may be an oil-in-water emulsion, a water-in-oil emulsion, or a water-in- oil-in-water emulsion.

EXPERIMENTAL EXAMPLES THE INVENTION WILL NOW BE DESCRIBED IN MORE DETAIL with reference to the following experimental examples and accompanying figures. In the figures: FIGURE 1 shows experimental formulations to determine physical properties of MagOil® in emulsion; 1% MagOil® emulsion (A), 4% MagOil® emulsion with testing different surfactants for emulsion creaming (B), Several hours later testing for visual creaming of emulsion (C); FIGURE 2 shows MagOil® emulsions (1 and 2%); FIGURE 3 shows 1 and 2% MagOil® emulsion at different time points during stability analysis; Day of manufacture (A), 2 weeks (B}, 6 weeks (C), 8 weeks (D}, 14 weeks (E); FIGURE 4 shows 4% MagOil® emulsion at different time points during stability analysis; day of manufacture (A), 4 weeks (B), 6 weeks (C), 12 weeks (D), 14 weeks (E), 26 weeks (F); FIGURE 5 shows 4% MagOil® emulsion with antioxidant at different time points during stability analysis; day of manufacture (A), 4 weeks (B), 6 weeks (C), 12 weeks (D), 14 weeks (E), 26 weeks (F);

FIGURE 6 shows 4% MagOil® emulsion with antioxidant and immunostimulant 1 (Saponin) at different time points during stability analysis; 2 weeks (A), 4 weeks (B), 6 weeks (C), 10 weeks (D), 12 weeks (E), 24 weeks (F); FIGURE 7 shows 4% MagOil® emulsion with antioxidant and immunostimulant 2 ( Lipid A analogue) at different time points during stability analysis; 2 weeks (A), 4 weeks (B), 8 weeks (C), 12 weeks (D); FIGURE 8 shows 4% MagOil® emulsion with antioxidant and immunostimulant 3 (Imidazoquinoline) at different time points during stability analysis; 2 weeks (A), 4 weeks (B), 8 weeks (C), 12 weeks (D);

FIGURE 9 shows particle size of 1 and 2% MagOil® emulsions over a 27 week stability period; DOM — Day of manufacture;

FIGURE 10 shows stability of 4% MagOil® and 4% MagQil® emulsion with antioxidant particle size over 14 weeks stability period; DOM — Day of manufacture; FIGURE 11 shows stability of 4% MagOil® emulsion with an immunostimulant particle size over 24 weeks; DOM — Day of manufacture;

FIGURE 12 shows stability of 4% MagOil® emulsion with an immunostimulant 2 (Lipid A analogue) particle size over 12 weeks; DOM — Day of manufacture;

FIGURE 13 shows stability of 4% MagOil® emulsion with an immunostimulant 3 (Imidazoquinoline) particle size over 12 weeks; DOM — Day of manufacture;

FIGURE 14 shows example graphs of the size distribution of particles size vs volume in the MagOil® emulsions with each reading in triplicate; 1% MagOil® emulsion 4°C (A), 4% MagOil® emulsion 4°C (B), 4% MagOil® emulsion with antioxidant 4°C (C), 4% MagOil® emulsion with immunostimulant 4°C (D);

FIGURE 15 shows cytotoxicity of MagQil® and squalene stable emulsions against murine RAW264.7 macrophages.

A, MagQOil® stable emulsion in the absence and presence of immunostimulants, Imidazoquinoline and Saponin; B, MagOil® and squalene stable emulsions in the absence and presence of immunostimulant Lipid A analogue; C, LPS as positive control (error bars represent the standard deviation of quadruplicate values);

FIGURE 16 shows TNF-a production by RAW264.7 macrophages following 24 h treatment with MagOil® and squalene stable emulsions in the absence and presence of immunostimulants (error bars represent the standard deviation of triplicate values and LPS was used as positive control);

FIGURE 17 shows IL-6 production by RAW264.7 macrophages following 24 h treatment with MagOil® and squalene stable emulsions in the absence and presence of immunostimulants (error bars represent the standard deviation of triplicate values and LPS was used as positive control);

FIGURE 18 shows IL-10 production by RAW264.7 macrophages following 24 h treatment with MagOil® and squalene stable emulsions in the absence and presence of immunostimulants (error bars represent the standard deviation of triplicate values and LPS was used as positive control);

FIGURE 19 shows CXCL2/MIP-2 production by RAW264.7 macrophages following 24 h treatment with MagOil® and squalene stable emulsions in the absence and presence of immunostimulants (error bars represent the standard deviation of triplicate values and LPS was used as positive control); and

FIGURE 20 shows Squalene stable emulsion compared with MagOil® emulsion for stability of the particle size over time.

Pharmaceutical excipient in accordance with the invention

In the examples that follow, the lipid composition that was used was one derived from insect tissue, specifically from raw larvae of the black soldier fly, in the form of an oil. More specifically, the lipid composition that was used was that which is produced by Agriprotein UK (Pty) Ltd under the brand name MagOil®.

Example 1: Investigating sustainable oil from insect larvae as an lipid component in oil emulsion adjuvant formulations for veterinary applications

1.1 Methods

1.1.1 Physiochemical properties MagOil® is solid at room temperature, especially when atmospheric temperatures are cooler. An initial experiment was undertaken to determine the effects of changing temperature on MagOil® before emulsions were manufactured. This was done to determine whether MagQil® would cause an emulsion thereof to solidify at the temperatures used for determining stability of the emulsion. Solidification of the formulation during manufacture needs to be avoided, as it would be detrimental to manufacturing equipment. Initial mixing was done using a high shear mixer. The resulting formulation can be seen in Figure (1).

The formulation was left to stand for a few hours to monitor solidification and separation of the emulsion. The stability of the emulsion was monitored over a few days at various oil percentages.

Once the formulation was deemed stable in liquid form, the next step was to complete emulsion formulation which included size reduction of the oil particles by microfluidization, as described in the emulsion formulation section below.

1.1.2 Emulsion formulation Basically, the same protocol was followed for the preparation of each emulsion described herein, with modifications as described. For each emulsion, an oil phase was prepared by adding 2-4% MagOil® and 1,9% (w/v) phosphatidylcholine, final concentration, to the mixture (either natural or synthetic) into a glass jar. Since MagOil® is solid at room temperature, it was heated to 30°C before starting the emulsion procedure. In certain formulations, an antioxidant in the form of alpha tocopherol (Vitamin E) was added to the emulsion to prevent any oxidation of the MagOil®. The aqueous phase consisted of ammonium phosphate buffer (24,3 mM ammonium phosphate monobasic, 1,3 mM ammonium phosphate diabasic, 258,4 mM glycerol) pH 5.5-5.8 and surfactant either Poloxamer or sorbitan mono-oleate at a final concentration of 0.36 mg/ml.

The aqueous phase was added at 90% (v/v) to the oil phase. The oil and aqueous phase were mixed with a heavy-duty laboratory mixer at approximately 3000 rpm for 10 min which yielded a crude emulsion.

This crude emulsion was then homogenised with a microfluidizer for 10 passes at approximately 25 000 psi. Completed formulations were filter sterilised though 0.22 um filters and monitored for stability over 3-6 months at 2-8°C, room temperature and 40°C.

Table 1. MagOil® emulsions formulated and developed in the Afrigen laboratories MagOil®, PC’ and surfactants to produce a pH and particle size 1 14 weeks stable oil in water emulsion analysis Visual appearance, MagQil®, PC’, surfactants and antioxidant to pH and particle size 2 26 weeks prevent oxidation of the product analysis Visual appearance, MagOil®, PC’, surfactants, antioxidant and pH and particle size 3 24 weeks immunostimulatory molecule 1 {Saponin) analysis MagQil®, PC’, surfactants, antioxidant and Visual appearance, 4 immunostimulatory molecule 2 (Lipid A pH and particle size 12 weeks analogue} and 3 (Imidazoguinoling) analysis

"PC phosphatidyicholine

1.1.3 Quality control parameters The formulated MagOil® emulsions were tested for stability by visual appearance of the emulsion, pH and particle size of the oil emulsion droplets. The emulsions were stored at three different temperatures 4, 20 and 40°C to see the effects on the emulsions. The 20 and 40°C stability temperatures were used for accelerated stability results and effects thereof.

1.1.3.1 Visual appearance The visual appearance of the emulsions was a white milky like liquid. The oil and aqueous phases remained intact with no separation of the phases over time. Emulsions were analysed over a 6 month period (24 weeks) and analyses were carried out every 2 weeks from the time of manufacture where possible.

1.1.3.2 pH The pH of the formulated emulsions needs to remain within physiological limits if the emulsion is to be used as a parenteral or injectable formulation and therefore the pH was analysed over time.

1.1.3.3 Particle size analysis

The emulsions formulated were nanoparticulate, ensuring better absorption and presentation to a host immune system. The particle size of the emulsion needs to be in the nano particle range of 90-110 nm to maintain the nanoemulsion.

In the present case, average particle size was determined using dynamic light scattering measurement for stability over time. For a solution containing homogeneous sized particles, one would expect a single peak.

1.2 Results Various emulsions were prepared with MagOil® as the oil component. This was completed to test the percentage of oil that could be tolerated in the emulsion. Solidification of the MagOil® in the emulsion formulation was tested in initial experiments, as described above. Initial formulations contained 1 and 2% MagOil® as oil component in a total of 100 mL emulsion. These were manufactured and submitted for stability at three different temperatures. Further percentage oils with other additives were later prepared, once the first set of stability results were obtained with the 1 and 2% emulsions (Table 1).

1.2.1 Visual appearance

1.2.1.1 1 and 2% MagOil® emulsions The 1% MagOil® emulsion was visually opalescent (Figure 2) when compared to stable emulsion made with squalene as oil component. However, the percentage MagOil® used was below the concentration (of oi!) used in squalene emulsions.

The 2% MagOil® emulsion was similar in appearance to a squalene emulsion. Stability tests were done over a 14 week period. Figure 2 shows the MagOil® emulsions after manufacture and filtration. Figure 3 shows the 1% MagOil® emulsion after 14 weeks.

The sample stored at 40°C was slightly yellow after 2 weeks at 40°C and remained this way for all stability analysis time points.

The emulsions in Fig. 2 and 3 were tested again at 27 weeks after manufacture.

Visually all of the emulsions were still intact however the particle size for both emulsions were variable and the pH were out of the physiological pH range (see Figure 8).

1.2.1.2 4% MagOil® without and with antioxidant A 4% MagOil® emulsion was manufactured which is comparable to the percentage oil used for stable emulsion containing squalene (Figure 4). The visual appearance was a white milky liquid which is what is expected.

A second batch of 4% MagOil® was also manufactured with an antioxidant to protect the oil and phospholipids in the emulsion. Once again, the batches were submitted to stability analyses over a 14- week period. When inspected visually, the emulsion appeared stable at all temperatures over 14 weeks.

The visual appearance of both the 4% MagOil® emulsion (Figure 4) and the 4% MagQil® emulsion with antioxidant (Figure 5) remained the same at 4 and 20°C with the samples at 40°C yellowing slightly.

The 4% emulsions were tested for visual stability at 26 weeks post manufacture. Visually the emulsions were still intact with no separation (Figure 4 F). The same can results were observed at 26 weeks stability analysis for the 4% emulsions with antioxidant added to the emulsion formulation (Figure 5 F).

1.2.1.3 4% MagOil® emulsion with immunostimulatory compound 1 (Saponin) 4% MagOil® emulsion with an immunostimulatory compound, Saponin, used widely in veterinary vaccine applications was manufactured. Figure 6 shows the stability of the formulation over a 22 week period and all remains in a visually stable emulsion formulation. The pH and particle sizes did show variation (see particle size results).

1.2.14 4% MagOil® emulsion with immunostimulatory compounds 2 and 3 (Lipid A analogue and Imidazoquinoline) 4% MagOil® emulsion with an immunostimulatory compound 2, Lipid A analogue, and compound 3, Imidazoquiniline, were manufactured specifically for comparison for bioactivity assays being completed on the MagOil® formulations.

These formulations were tested for stability as the other formulations were however, with shorter stability data available. Below Figure 7 and 8 shows the stability of the two formulations over the last 12 weeks. The Lipid A analogue has remained stable visually over the past 12 weeks.

The MagOil® emulsion with Imidazoguinoline was visually stable for the first 4 weeks at all temperature points, however after 4 weeks the sample stored at 40°C started showing instability with the oil phase separating from the emulsion (Figure 8 C and D) this is accelerated stability and these results are expected. The samples stored at 4 and 20°C has remained visually intact for the past 12 weeks which is acceptable for the formulation.

1.2.2 pH The pH of the formulations was analysed for stability over the same time period. Table 2 gives a summary of the pH of the emulsions stored at 4°C on day of manufacture until 27, 24 and 12 weeks for the various formulations. The pH of samples at 20°C and 40°C were variable between the emulsions over time. At the 10, 12 and 14 week stability test the accelerated samples pH had declined below the acceptable levels which are between 5.6 and 5.8.

Table 2. pH analysis of MagOil® emulsions at 12 week, 24 week and 27 week samples stored at 4°C samples ee eo OO TE Megol emisonddeg Tees 2% MagQil® emulsion 4 deg T= 27 weeks 5,67 4% MagQil® emulsion 4 deg? T= 26 weeks 5,73 4% MagOil® emulsion + antiox 4 deg? T= 26 weeks 5,72 4% MagQil® emulsion immunostimulant Saponin 4 T= 24 weeks 5.70 deg? 4% MagQil® emulsion immunostimulant (Lipid A T= 12 weeks 5.60 analogue and Imidazoquinoline) 4 deg

2 emulsions at 22-24 weeks; ® emulsion currently at 12 weeks.

1.2.3 Particle size Particle size was analysed for all the emulsions, and the analysis is presented in the graphs and tables referenced. See Example 3 and Figure 20 for a comparison of MagOil® stability versus stability of an emulsion manufactured with shark derived squalene used currently for adjuvant manufacture.

Table 3. Particle size analysis with Z-average (d.nm) 2 standard deviation for 1 and 2% MagOil® emulsions pg —...,LL___ —. MagOil® 9517 £1,9 4°C 90,88 + 1,1 90,08 + 1,2 90,39 + 0,6 89,82 + 0,7 90,75+1,3 91,6124 20°C 94,36 +3 91,76 £1,9 92,5911,8 97,95+28 117, +124 110,1 £45 40°C 112,0711,9 2188339 2555379 291,77+6,7 139,1 +21 188,1 + 1,35 2% MagOii® 95,17 +2,7 4°C 91,4311,9 91,2611,2 91,21+1,9 91,43+1,3 90,33 + 1,1 91,1207 20°C 91.51 £15 91,98+1,7 92,34 £21 92,97 11,3 91,47 £1 94,28+1,7 40°C 93,94 11,4 135,43+2,8 14043128 189,23+2,8 128,57 £2 556 + 13,4 =Day of manufacture

The stability at 40°C for both 1 and 2% MagOil® formulations increased over time or was variable. This is expected at the accelerated temperature stability samples.

Table 3 and Figure 8 show the particle size stability of the 1 and 2% emulsions at three temperature storage conditions. lt is clear that the MagOil® emulsions stored at 40°C are not as stable as those stored at 4 and 20°C after 27 weeks. The 4% MagOil® emulsions with and without antioxidant performed better, with the particle size remaining the same after the 26 week stability period (Table 4 and Figure 9).

Table 4. Particle size analysis with Z-average (d-nm) + standard deviation for 4% emulsions with and without antioxidant ce 4°C 94,922 94,6 +2 94.3 +1 94 +04 91,9 +1 94,7 11,7 20°C 96,113 96,7 12 95,4 +1 94,9 £1 93,9 £1 96x15 40°C 98,2 +2 96,312 96,4 = 1 97.9% 1 97,9 +1 208,6 +3,2 4% MagOil® with antioxidant 91,79 £1 4°C 93,1+3 92 +2 92 +2 91,6+2 90,6 + 1 91,7 11,4 20°C 92,91+2 93.312 93,3+1 926+2 91,2+0,2 92,9 11,7 40°C 96,513 101,22 101,2£2 104,4 +2 105,5+1 919411 “ay of marvtoctoe Table 5. Particle size analysis with Z-average (d-nm) x standard deviation for 4% emulsions with immunostimulant 1 (Saponin} a Os with immunostimulant 1 (Saponin) 91,79 £1 4°C 1006 +2 99,3+2 99,5 £1 99,4 £1 99,2 £1 101,1£1,3 101,415 20°C 101,51 100,2 +1 102+ 2 100,52 100,4 £1 101,89 41,7 105,8 + 8,1 40°C 1008 +2 100,5+2 104 £2 103.4 £1 108.2 £1 113415 nm aDay ddmanutacture; nm- not measured

The particle sizes for the 4% MagOil® with immunostimulant 1 (Saponin) has shown to be stable over 24 weeks at 4 and 20°C (Table 5 and Figure 11). The emulsion at 40°C was out of specification for particle size and was therefore not measured at 24 week stability.

Only samples that showed favourable pH profiles were included in the particle size stability analysis.

Further formulations were manufactured to include for the bioactivity assays.

These formulations where MagQil® with specific immunostimulants (Lipid A analogue and an Imidazoquinoline). These MagOil® formulations would be directly compared to stable emulsion with shark squalene as oil component, with the same immunostimulants.

The particle sizes for the 4% MagOil® with immunostimulant 2 (Lipid A analogue) was shown to be stable over 12 weeks at all temperatures (Table 6 and Figure 12). The MagOil® emulsion with immunostimulant 3 (Imidazoquinoline) has been stable over the past 12 weeks at 4°C and 20°C.

The emulsion at 40°C was out of specification for particle size and was therefore not measured at 12 week stability (Table 6 and Figure 13).

Table 6. Particle size analysis with Z-average (d-nm) x standard deviation for 4% emulsions with immunostimulant 2 {Lipid A analogue) and 3 (Imidazoquinoline) Maa WE Sea ua “4% Magoil@ wi immunostmulant 2 (Lipid Aanslogue) 9 4°C 9122 91,3£1.2 91.5 +1 91.4 £1 20°C 91,321 91,817 92:18 93,8 £1 40°C 92,8 20,8 99,1+1,8 116 £1 93,7 10,8 4% MagOil® with immunostimulant 3 (Imidazoguinoiine) 107+0,8 4°C 106,5 + 0,6 107,5+1,8 107,3+2,2 1055+1,5 20°C 107,5 + 1,3 108,8 £1,8 107,4 +2 107,7 £0,4 40°C 137,5+1,8 218,2+3,5 183,6 +35 nm om- not measured

Table 7. Summary of particle size analysis at day of manufacture and current stability point of emulsions stored at 4°C. 10303535 „ere esin Polydispersity’ (nm T=12- Polydispersity?

I TE 2% MagOil® emulsion 95,27 + 2,68 0,17 + 0,005 90,33 + 1,05 0,15 £ 0,04 4% MagQil® emulsion 93,76 10,9 0,13 + 0,004 91,9+1,1 0,13 +0,013 4% MagOil® emulsion + antioxidant 91,7911,0 0,13 £0,009 90,6 £ 0,9 0,13:t 0,005 4% MagOil® emulsion immunostimulant 1 (Saponin) 91,7911,0 0,13 £0,009 99,2110.8 0,14 + 0,008 4% MagOil® emulsion immunostimulant 2 (Lipid A analogue) 107,2+0,1 0,11 £0,02 107,7 £0,4 0,1 10,02 4% MagQil® emulsion immunostimulant 3 {(Imidazoguinoline) 89,7 £0,1 0,11 + 0,01 93,7 £0,8 0,1 + 0,01 sPolydispersity Index indicates the width of the overall distribution, assuming a single mean, and it ranges from 0 to 1. Pdi values between 0.08 and 0.7 is the mid-range value of polydispersity. Pdl values higher than 0.7 indicates a very broad distribution of particle sizes and is not desirable.

1.3 Discussion Emulsions formulated with MagOil® were found to be stable for over 3 months. The 4% MagOil® emulsions were found to be the most stable.

Example 2: Investigating in vitro biological activity of stable emulsions comprising oil from insect larvae as a lipid component

2.1 Introduction MagQil® emulsions have been shown to be stable at 4°C with reference to pH, particle size analysis and visually. The next phase in the development of adjuvant formulations containing MagOil®, as the oil component, was to complete jn vitro biological activities with the formulated MagOil® emulsions with and without immunostimulants. MagOil® emulsions were compared to shark derived squalene stable emulsion (SE) with and without immunostimulants currently being used in various clinical trials.

Cytokines are small, water-soluble proteins secreted by various cell types, including immune cells (e.g. macrophages, mast cells, B- and T lymphocytes), endothelial cells, fibroblasts and stromal cells. Cytokines are intercellular messengers which modulate humoral immune (i.e. antibody production), innate immune (i.e. cell-based) and inflammatory responses, and regulate the maturation, growth and responsiveness of cell populations. Cytokines are divided into lymphokines, chemokines, interleukins and monokines.

Cytokine secretion is an indication of inflammation in humans and animals. Macrophages, exposed to inflammatory stimuli, will secrete pro-inflammatory cytokines [e.g. tumour necrosis factor (TNF), interleukin (IL)-1, IL-6, IL-8 and IL-12] to recruit other immune cells. Inflammation is regulated by multiple inhibitors and antagonists, e.g. anti-inflammatory cytokines [incl. IL-10 and transforming growth factor (TGF)-B]. Chemokines are involved in the cellular migration (process:

chemotaxis) of immune cells to the source of secretion (i.e. macrophages). Pro-and anti-inflammatory cytokines are associated with Th1 and Th2 immune responses, respectively. Squalene SEs are associated with Th1 immune responses, whereas immune responses of MagQil® SEs are unknown.

The aim of the study was to determine and compare the in vitro biological activities — cytotoxicity and cytokine secretion of MagOil® and squalene stable emulsions, in the absence and presence of immunostimulants, against murine RAW264.7 macrophages. RAW264.7 macrophages were chosen as cell line because the MagOil® stable emulsions will be tested in mice for safety and efficacy. The objectives of the study included:

1. To determine and compare the cytotoxicity of MagOil® and squalene stable emulsions, in the absence and presence of immunostimulants, against RAW264.7 macrophages.

2. Todetermine and compare the effect of MagOil® and squalene stable emulsions, in the absence and presence of immunostimulants, on pro-inflammatory cytokine [i.e. tumour necrosis factor (TNF)-a and interleukin (IL)-6] secretion in RAW264.7 macrophages.

3. Todetermine and compare the effect of MagOil® and squalene stable emulsions, in the absence and presence of immunostimulants, on anti-inflammatory cytokine (i.e. IL-10) secretion in RAW264.7 macrophages.

4. Todetermine and compare the effect of MagOil® and squalene stable emulsions, in the absence and presence of immunostimulants, on chemokine Île.

CXCL2/macrophage inflammatory protein (MIP-2)] secretion in RAW264.7 macrophages.

2.2 Cell Viability and Cytotoxicity The viability of RAW264.7 macrophages was used to determine cytotoxicity of MagOil® stable emulsions, in the absence and presence of immunostimulants.

2.2.1 Methods RAW264.7 macrophages were routinely maintained in complete culture medium containing RPMI 1640 medium, 10% foetal bovine serum and penicillin-streptomycin in culture dishes. AW264.7 macrophages were seeded in 96-well plates at cell densities of 20 000 cells/100 uL/well. MagOil® stable emulsion (MG-SE) was diluted in complete medium to reach final MagOil® percentages of between 0,025 and 0,5%. Shark squalene stable emulsion (SE) was diluted with complete medium to reach final squalene percentages of between 0,02 and 0,4%. These negative control dilutions corresponded to the final oil percentages in stable emulsions, containing immunostimulants, when diluted.

MG-SEs, containing Imidazoquinoline and Saponin, were diluted with complete medium to reach final immunostimulant concentrations of between 1,25 and 25 pg/mL. MG-SE and SE, containing Lipid A analogue, were diluted with complete medium to reach final immunostimulant concentrations of between 0,18 and 3,52 ug/mL. Cel! viability was determined using the 3-(4,5-dimethyl-2-thiazolul)-2,5-diphenyl-2H-

tetrazolium bromide (MTT) assay as described by Holst-Hansen and Brunner (1998) and the absorbance read at 540 nm using a BioTek® PowerWave XS spectrophotometer. The following formula was used to determine viability: Viability (%) = Absorbance of treated cells/absorbance of untreated cells x 100 RAW264.7 macrophage viability was used to determine the MagOil® and squalene percentages, and immunostimulant concentrations at which RAW264.7 macrophages should be treated for cytokine secretion.

2.2.2 Results and Discussion MagOil® and squalene percentages in MG-SE and SE, respectively, corresponded to the final oil percentages in the immunostimulant containing stable emulsions when diluted. SE (>0,04%) was more cytotoxic against RAW264.7 macrophages than MG- SE (>0,5%). The percentage (0,1-0,5%) of MagOil® was ~10 times higher than the percentage (0,01-0,04%) of squalene in the stable emulsions (Figure 15A and B). For in vitro studies three immunostimulants were formulated with MG-SE for comparison. MG-SEs, containing Imidazoquinoline and Saponin, were more cytotoxic than the MG- SE negative control, whereas MG-SE containing Lipid A analogue was not cytotoxic against RAW264.7 macrophages. MG-SE containing Lipid A analogue was less cytotoxic against RAW264.7 macrophages than SE containing Lipid A analogue (Figure 15B). Results of the cytotoxicity based on immunostimulants was: Imidazoquinoline>Saponin>Lipid A analogue. LPS was not cytotoxic (even at the highest concentration: 500 pg/mL)

against RAW264.7 macrophages (Figure 15C). LPS was used as a positive control for cytokine production; hence, it was important to determine the non-cytotoxic concentrations.

2.3 Cytokine Production The pro-inflammatory cytokines, anti-inflammatory cytokine and chemokine investigated were TNF-a and IL-6, IL-10 and CXCL2/MIP-2, respectively.

2.3.1 Method RAW264.7 macrophages were routinely maintained in complete culture medium containing RPMI 1640 medium, 10% foetal bovine serum and penicillin-streptomycin in culture dishes. RAW264.7 macrophages were seeded in 12-well plates at cell densities of 200 000 cells/mL/well.

From the cytotoxicity results (Figure 14), three concentrations for each treatment were chosen: MG-SE (0.1, 0,2 and 0,5%; negative control), MG-Imidazoquinoline-SE (0,31, 0,62 and 1,25 pg/mL), MG-Saponin-SE (2,5, 5, 10 ug/mL), SE (0,01, 0,02, 0.04%; negative control), Lipid A analogue-SE (0,09, 0,18 and 0,35 pg/mL), MG-Lipid A analogue-SE (0,88, 1,76 and 3,52 pg/mL) and LPS (50 and 500 ng/mL; positive control). The following kits were purchased from R&D Systems with protocols available online:

1. Mouse TNF-a

2. Mouse IL-6

3. Mouse IL-10

4. Mouse CXCL2/MIP-2

2.3.2 Results and Discussion

2.3.2.1 TNF-a.

Mouse TNF-a concentrations in the undiluted samples were determined from a TNF- a standard curve (R? = 0,9822, slope: y = 0,0049 x) ranging between 10,9 and 700 pg/mL. For the MG-SE, MG-Imidazoquinoline-SE, MG-Saponin-SE, MG-Lipid A analogue-SE, Lipid A analogue-SE and LPS treatments, the optical density (OD) readings were above the maximum absorbance levels (i.e. overflow); hence these treatment- and TNF-a standard wells were diluted five times with ddH20 and the absorbance measured. The diluted standard curve’s slope (y = 0,001 x; R2 = 0,9834) was five times less than the original standard curve’s slope; hence it was assumed that TNF-a concentrations in the diluted samples were a true reflection of TNF-a concentrations in the undiluted samples. TNF-a levels were elevated (approx. four times) in all the treatments except for 0,01 and 0,02% SE where the TNF-a levels corresponded to the baseline (i.e. untreated cell control) (Figure 16). 2322IL-6 Mouse IL-6 concentrations in the undiluted samples were determined from an IL-6 standard curve (R?=0,9971, slope: y = 0,0051 x) ranging between 7,8 and 500 pg/mL. For the MG-SE, MG-Saponin-SE, MG-Lipid A analogue-SE and LPS treatments, the OD readings were above the maximum absorbance levels (i.e. overflow); hence these treatment- and IL-6 standard wells were diluted five times with ddH20 and the absorbance measured. The diluted standard curve’s slope (y = 0,0011 x; R? = 0,9976) was five times less than the original standard curve’s slope; hence it was assumed that IL-6 concentrations in the diluted samples were a true reflection of IL-6 concentrations in the undiluted samples. IL-6 levels increased dose-dependently for all treatments, except for SE where the IL-6 levels corresponded to the baseline (untreated cell control; Figure 17). The lower IL-6 level at the highest MG-SE percentage (0,5%) might be attributed to cytotoxicity. Saponin and Lipid A analogue were more effective in activating IL-6 secretion than Imidazoquinoline. This might be attributed to different innate immune system signalling pathways being activated. Cell type might also influence IL-6 production/secretion. 2323IL-10 Mouse IL-10 concentrations in the undiluted samples were determined from an IL-10 standard curve (R2 = 0,994, slope: y = 0,0023 x) ranging between 15,6 and 1000 pg/mL. None of the formulations have shown any anti-inflammatory activity. Based on IL-10 levels, MagQil®, squalene or the immunostimulants tested had no anti- inflammatory activity (Figure 18). LPS is a pro-inflammatory immunostimulant; hence it was not a suitable positive control to use. The internal IL-10 standard confirmed that the IL-10 ELISA kit worked optimally.

2.3.2.4 CXCL2/MIP-2 Mouse CXCL2/MIP-2 concentrations in the undiluted samples were determined from a CXCL2/MIP-2 standard curve (R? = 0,9903, slope: y = 0,0035 x) ranging between 7,8 and 500 pg/mL. All the OD readings were above the maximum absorbance levels (i.e. overflow); hence treatment- and CXCL2/MIP-2 standard wells were diluted five times with ddH20 and the absorbance measured. The diluted standard curve’s slope (y = 0,0007 x; R? = 00,9894) was five times less than the original standard curve’s slope; hence it was assumed that CXCL2/MIP-2 concentrations in the diluted samples were a true reflection of CXCL2/MIP-2 concentrations in the undiluted samples. Imidazoquinoline was more effective in inducing CXCL2/MIP-2 secretion than Saponin and Lipid A analogue (Figure 19). The in vitro biological activities of MagOil® and squalene SEs are summarised in Table

8.

Table 8. Comparison summary between MagOil® and squalene SEs of the in vitro biological assays performed on RAW264.7 macrophages.

U Cytotoxieiy CO ICxw002004% TNF-a secretion +++ ++ IL-6 secretion ++ - IL-10 secretion - - CXCL2/MIP-2 secretion +++ +++ *Percentage of MagOil® in SE were 10X higher than percentage of squalene in SE tested (based on cytotoxicity). Cytokine secretion levels: high (+++), medium (++), low {+}, baseline (-).

2.4 Conclusion The following points summarise the biological activities of MagOil® stable emulsions, in the absence and presence of immunostimulants, against RAW264.7 macrophages:

1. MagOil® stable emulsion was less cytotoxic than squalene stable emulsion against RAW264.7 macrophages.

2. The Imidazoquinoline was more cytotoxic than Saponin and Lipid A analogue when formulated in a MagOil® stable emulsion.

3. MagQOil® induced pro-inflammatory cytokine (i.e. TNF-a and IL-6) and chemokine (i.e.

4. CXCL2/MIP-2) secretion in RAW264.7 macrophages in the absence and presence of immunostimulants.

5. MagGil® did not induce anti-inflammatory cytokine (i.e. IL-10) secretion in RAW264.7 macrophages in the absence and presence of immunostimulants. Example 3: Comparative stability results between a 4% MagOil® emulsion and a 4% squalene stable emulsion Tables 9 to 11 and Figure 20 below are for comparative stability results between a 4% MagOil® emulsion and a 4% squalene stable emulsion. The squalene stable emulsion stability, Table 8, was followed for 12 months at 4°C with the 20°C and 37°C samples only being measured until 3 months as they were outside of the stability specifications. the MagOil® accelerated stability temperature is 40°C in line with industry best practices. Table 9. Particle size analysis with Z-average (d-nm) 2 standard deviation for 4% MagOil® emulsions 4% MagOil® (4°C) 94,9 +2 94,6 +2 94,3 +1 94 +0,4 91,9 £1 4% MagOil® (20°) 96.1 £3 96,7 +2 95.4 +1 94,9 +1 93,9 +1 4% MagOil® (40°) 98212 96,3+2 96,4 £1 97,9 + 1 97,9 + 1 =DOM- Day of manufacture

Table 10. Particle size analysis with Z-average (d.nm) t standard deviation of 4% stable emulsion manufactured with squalene me SE 003 (4°C) 103.7+1.6 1036+0.8 103.6+1.02 1056+232 101.2+1.45 SE 003

102.9:£1.4 1029+0.72 109.1 £0.65 (20°C) nm’ nm! SE 003 151 £15 268,4% 100 320 + 83 (37°C) nm! nm’ 2DOM- Day of manufacture 'nm-not measure The manufacture of the squalene emulsion and MagOil® emulsion was identical except for the surfactant used in the squalene final formulation which differs. In Figure 13, the particle size stability of the squalene emulsion against the MagOil® emulsion is shown. At 4°C and 20°C the stability is very similar in both the emulsions. The particle sizes vary slightly between the two emulsions which is expected as the two oil excipients are different. Also, the combination of excipients could also affect the particle size of the final emulsion. Particle size specifications are between 90-110 nm. In order for the MagOil® emulsion to pass stability it has to compare favourably to the squalene stable emulsion. The squalene emulsion stability for particle size and pH was used as a baseline for the MagOil® emulsions. The MagOil® emulsion stability was followed more closely, every two weeks, compared to the squalene since MagOil® is a novel excipient.

As is shown in the results the 4% MagOil® used in the manufacturing of an oil in water emulsion are more stable at 4°C and 20°C than the 1 and 2% MagOil® emulsions. pH stability of 4% squalene stable emulsion over a 1 year period is shown in Table 9 to compare to the MagOil® emulsion pH in Table 2. Table 11. pH stability of squalene stable emulsion formulation over 1 year period. == SE 003 (4°C) 5.73 5.73 5,82 5,8 SE 003 (20°C) 5.72 5.6 nm’ nm! SE 003 (37°C) 5.36 4.32 nm! nm! 2DOM- Day of manufacture inm-not measure

Claims (9)

CONCLUSIONS A pharmaceutical adjuvant composition comprising an emulsion of a lipid composition, in the form of an oil derived from insect tissue, in water; and a pharmaceutical adjuvant, in the emulsion. The pharmaceutical adjuvant composition of claim 1, wherein the insect tissue from which the lipid composition is derived is crude larvae of the black soldier fly (Hermetia illucens). A pharmaceutical composition comprising a pharmaceutical excipient comprising an emulsion of a lipid composition, in the form of an oil derived from insect tissue, in water; and a therapeutic or prophylactic agent. The pharmaceutical composition of claim 3, wherein the insect tissue from which the lipid composition is derived is crude larvae of the black soldier fly (Hermetia illucens). A pharmaceutical composition according to claim 3 or claim 4, wherein the pharmaceutical excipient is a pharmaceutical excipient composition according to claim 1 or claim 2. A pharmaceutical composition according to any one of claims 3 to 5, wherein the therapeutic or prophylactic agent is a vaccine or immunomodulator. NL31175-HS The pharmaceutical adjuvant composition of claim 1 or claim 2 for use in a method of enhancing or promoting an immune response in a patient, wherein the immune response is stimulated by administration of a vaccine, immunomodulator or other therapeutic agent to the patient . Use of a lipid composition, in the form of an oil derived from insect tissue, in the manufacture of a pharmaceutical adjuvant composition for enhancing or promoting an immune response in a subject, wherein the immune response is stimulated by administration of a vaccine, immunomodulator or other therapeutic agent for the subject. Use according to claim 8, wherein the pharmaceutical adjuvant composition is a pharmaceutical adjuvant composition according to claim 1 or claim 2. NL31175-HS