Safety evaluation of a glutaraldehyde modified tyrosine adsorbed housedust mite extract containing monophosphoryl lipid A (MPL®) adjuvant: a new allergy vaccine for dust mite allergy

Vaccine 20 (2002) 737–743

Safety evaluation of a glutaraldehyde modified tyrosine adsorbed housedust mite extract containing monophosphoryl lipid A (MPL®) adjuvant: a new allergy vaccine for dust mite allergy Paul Baldrick a , Derek Richardson b,∗ , Alan W. Wheeler b a

Covance Laboratories Ltd., Otley Road, Harrogate, North Yorkshire HG3 1PY, UK Allergy Therapeutics Ltd., Dominion Way, Worthing, West Sussex BN14 8SA, UK

b

Received 19 February 2001; received in revised form 4 September 2001; accepted 20 September 2001

Abstract A new allergy vaccine is currently under clinical evaluation for the prevention or relief of symptoms caused by specific housedust mites. It consists of a 50:50 mixture of the mite Dermatophagoides pteronyssinus and D. farinae protein derived from aqueous extracts of the mites which is chemically modified by glutaraldehyde and adsorbed onto l-tyrosine with addition of the immunostimulatory adjuvant, monophosphoryl lipid A (MPL® ) “Polymite”. A specific preclinical safety testing strategy was developed to support clinical use and comprised single and repeat dose toxicity, reproduction toxicity and local tolerance studies. Dose levels of up to 0.5 ml for the mouse and up to 1 ml for both the rat and the rabbit were used. Overall, the product was shown to produce no toxicological findings of significance at levels greatly in excess to those proposed for clinical use. A not unexpected, but relatively minor, immunostimulatory effect was seen following repeated dosing (once weekly for 13 weeks) at 1 ml per rat; the Polymite formulation also resulted in injection site reaction which can largely be attributed to the presence of tyrosine. No reproduction toxicity was found. © 2001 Elsevier Science Ltd. All rights reserved. Keywords: Polymite; MPL® adjuvant; Allergy vaccine; Toxicity studies

1. Introduction Allergy vaccination (AV) is currently recognised as a safe and efficacious treatment strategy for the care of allergic asthma and allergy related disorders which are becoming increasingly prevalent in the developed world [8,16,25,26,30]. The introduction of measures to improve safety include optimal dosing, allergen modification (to reduce allergenicity whilst maintaining immunogenicity) and adjuvant adsorbtion (to control release). Polymite represents a new allergy vaccine with these properties for the prevention or relief of symptoms caused by specific housedust mites. This product is a 50:50 mixture of the mite Dermatophagoides pteronyssinus and D. farinae extracts which is chemically modified by glutaraldehyde and adsorbed onto l-tyrosine with addition of the immunostimulatory adjuvant, monophosphoryl lipid A (MPL® ). Treatments with allergen modification and improved adsorbtion are not new and include the modified pollen ∗ Corresponding author. Tel.: +44-1903-844700; fax: +44-1903-844744. E-mail address: [email protected] (D. Richardson).

allergens tyrosine adsorbate (MATA) vaccines derived from extracts of one or more pollens which are chemically modified by glutaraldehyde and adsorbed onto l-tyrosine. Variants of the MATA range are currently marketed in various countries and have been in use for at least 25 years. An array of data are available to show the safe and efficacious use of these materials [3,7,14,19,22,27]. Various publications show that l-tyrosine has a role as an immunological adjuvant [23,40,42]. The addition of MPL® adjuvant to a range of vaccine materials is the subject of ongoing clinical trials for many companies. Published non-clinical and clinical data indicate MPL® adjuvant to be safe and effective with excellent immunological adjuvant activity [1,15,21,36,37]. Dust mite allergy is a major problem world-wide and D. pteronyssinus has been indicated in perennial allergic rhinitis [2], atopic eczema [9] and asthma [33]. A role for D. farinae, which has a very similar skin prick test reactivity to D. pteronyssinus and demonstrates cross-antigenicity, has also been discussed in the literature [24]. Indeed, sensitisation to both D. pteronyssinus and D. farinae has been demonstrated in patients with asthma and rhino-conjunctivitis [13]. Preparations with D. pteronyssinus extracted proteins, adsorbed onto tyrosine, have been evaluated clinically

0264-410X/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S 0 2 6 4 - 4 1 0 X ( 0 1 ) 0 0 4 1 3 - 3

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with positive results [2,4,9,12,17,20,28,39]. However, some workers have also reported inconclusive results [29]. Recently, AV evaluation using house mite extracts has been shown to be safe and effective in asthmatic subjects [6,10]. Polymite is currently undergoing clinical trials and this paper reports preclinical studies that were performed to show that the material has no obvious toxicological safety issues. Relatively little regulatory documentation exists to give guidance for safety testing of these types of products. The World Health Organisation (WHO) has designated standardised allergen extracts as “vaccines” in a recent position paper on allergen immunotherapy [4]. In Europe, guidance exists for non-clinical evaluation of vaccines [5]. This guidance has been followed but due to the nature of this allergoid material, a specific safety testing strategy which was discussed with the Paul Erlich Institute, was adopted. Studies comprised single and repeat dose toxicity, reproduction toxicity and local tolerance studies. In order to mimic the clinical situation subcutaneous dosing was selected as the route of administration for these studies. Additionally, the intraperitoneal route was used in some single dose studies to examine any potential effects following maximal systemic exposure.

2.1. Test material and formulation Polymite is an equal part mixture of glutaraldehyde modified D. pteronyssinus and D. farinae allergoids [41] adsorbed onto l-tyrosine with the addition of MPL® adjuvant. Although a measured protein value is used for the starting material which forms the basis for the calculation of potency for the final product, assays for the major allergens Der p1 and Der f1 are also undertaken on the starting materials and on the freeze dried intermediates as a measure of biological activity. In addition immunoreactivity assays are carried out on the final product. These potency measurements are based on the reactivity of human IgG antibody with the modified allergens. The component parts of the product are described in Table 1 and of the control material in Table 2. The test material for all the safety studies was formulated into vials containing 10 ␮g/ml protein, 50 ␮g/ml MPL® adjuvant and 20 mg/ml tyrosine and was administered as supplied. The control material for the toxicity studies was a tyrosine blank formulation of 20 mg/ml (2%) tyrosine in 0.5% phenol in neutral buffered saline. For the local tolerance study, the control materials were of 20 mg/ml tyrosine blank formulation + 0.05 mg/ml MPL® adjuvant and physiological saline.

2. Materials and methods 2.2. Single dose toxicity studies All studies were performed at Covance Laboratories Ltd., Harrogate, UK and conformed to Good Laboratory Practice (GLP).

In these studies, groups of five male and five female CD-1 mice or Wistar rats were given a single subcutaneous or

Table 1 Composition of finished product Polymite Ingredient

Function

Active substances Allergen extract (D. pteronyssinus and D. farinae)

Active ingredient

Other ingredients l-tyrosine Monophosphoryl lipid A/AF (MPL® ) Phenol Sodium chloride Disodium phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate Water for injections a

Adsorbant base Adjuvant Preservative Tonicity Buffering and tonicity Diluent

Quantity per ml

Reference to standards

0.1, 1.0, 5 and 10 ␮g/ml protein

HSEa

20 mg 0.05 mg 5.0 mg 9.0 mg Not exceeding 4.0 mg

Ph.Eur. HSE Ph.Eur. Ph.Eur. Ph.Eur.

1.0 ml

Ph.Eur.

HSE: in-house specifications are measured in SU: standardised units/ml. Excipients such as phenol were present in both the control and test materials.

Table 2 Composition of the control material Ingredient

Function

Quantity per ml

Reference to standards

l-tyrosine Phenol Sodium chloride Disodium phosphate dodecahydrate and sodium dihydrogen phosphate dihydrate Water for injections

Adsorbant base Preservative Tonicity Buffering and tonicity

20 mg 5.0 mg 9.0 mg Not exceeding 4.0 mg

Ph.Eur. Ph.Eur. Ph.Eur. Ph.Eur.

Diluent

1.0 ml

Ph.Eur.

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intraperitoneal dose of Polymite at dose volumes of 0.05 and 0.5 ml per mouse or 0.1 and 1 ml per rat. Similarly sized control groups were also included for each treatment regimen, using the higher dose volume. At dosing, mice were approximately 6–7 weeks old and weighed 24–34 g (males) and 20–30 g (females) and rats were approximately 10–11 weeks old and weighed 253–312 g (males) and 179–207 g (females). All animals were killed after 2 weeks and underwent a full necropsy.

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signs, bodyweights and food consumption. Following sacrifice, all animals were examined for macroscopic structural or pathological changes, pregnancy status, gravid uterus weight, number of corpora lutea, number and intrauterine position of implantation sites and status of implantations (early/late intrauterine deaths, live/dead foetuses). Foetal assessment comprised external examination, foetal weight, placental weight, foetal sex, macroscopic (visceral abnormalities) and skeletal examination and examination for malformations.

2.3. Repeat dose toxicity study This study took the form of a weekly subcutaneous dose over a 13-week period in the rat followed by a 4-week treatment-free period. Groups of up to 20 male and up to 20 female Wistar rats were given a single subcutaneous dose of Polymite at dose volumes of 0.1 and 1 ml per rat once weekly for 13 weeks. A control group of 20 males and 20 females was also dosed at a dose volume of 1 ml per rat. Rats were approximately 7 weeks old and had mean bodyweights of approximately 160 g (males) and 126 g (females) on study initiation. All rats dosed with 0.1 ml and 10 animals/sex/group from the control and 1 ml groups were sacrificed after 13 weeks; remaining rats were retained for a 4-week recovery period. The following study parameters were measured: mortality and clinical observations—daily, bodyweights and food consumption—weekly, ophthmaloscopy (control and high dose)—pre-dose and week 12, urinalysis parameters—week 12 and haematology and clinical chemistry parameters— week 13. At study termination, rats were subjected to a complete necropsy after the treatment or recovery periods and selected organs weighed. A complete set of tissues/organs was collected from each rat and processed for microscopic examination for control and high dose group animals. Details on measured in-life, terminal and microscopic parameters are not reported but conformed to standard internationally accepted regulatory guidelines. 2.4. Reproduction toxicity studies Three investigations were performed—embryo-foetal development studies in the rat and rabbit and a pre- and post-natal development study in the rat. 2.4.1. Embryo-foetal study in the rat Groups of 24 female pregnant Sprague–Dawley rats were given a single subcutaneous dose of Polymite at dosage levels of 0.1 and 1 ml per animal on days 1, 6, 10, 14 and 17 of gestation. A control group of 24 females was also included at a dose volume of 1 ml per rat. Female rats were approximately 9 weeks of age and 201–278 g in weight at mating. Following the dosing period, dams were maintained to day 20 of gestation when they were killed and their uterine contents examined. Dams were observed for mortality, clinical

2.4.2. Embryo-foetal study in the rabbit Groups of 24 female pregnant New Zealand White rabbits were given a single subcutaneous dose of Polymite at dosage levels of 0.1 and 1 ml per animal on days 2, 7, 13, 19 and 24 of gestation. A control group of 24 females was also included at a dose volume of 1 ml per rabbit. Female rabbits were approximately 4–5 months of age and 3.1–4.0 kg in weight at mating. Following the dosing period, dams were maintained to day 29 of gestation when they were killed and their uterine contents examined. Dams were observed daily for mortality, clinical signs, bodyweights and food consumption. Following sacrifice, all animals were examined for macroscopic structural or pathological changes, pregnancy status, gravid uterus weight, number of corpora lutea, number and intrauterine position of implantation sites and status of implantations (early/late intrauterine deaths, live/dead foetuses). Foetal assessment comprised external examination, foetal weight, placental weight, foetal sex, macroscopic (visceral abnormalities) and skeletal examination and examination for malformations. 2.4.3. Pre- and post-natal development study in the rat Groups of 24 female pregnant Sprague–Dawley rats were given a single subcutaneous dose of Polymite at dosage levels of 0.1 and 1 ml per animal on days 1, 6, 10, 14 and 17 of gestation. A control group of 24 females was also included at a dose volume of 1 ml per rat. Female rats were 8–10 weeks of age and at least 160 g in weight at mating. The females were allowed to litter and rear their offspring to weaning. Twenty animals/group/sex were randomly selected for the F1 generation and were maintained untreated for at least 12 weeks post-weaning (maturation phase) before being paired for 15 days. The F1 mated females were killed on day 13 of gestation for examination of uterine content. The F1 males were killed in week 17. Clinical observations, bodyweights and food intakes of the parent animals were recorded as well as the physical and functional development of the offspring. For the F1 generation, clinical observations, bodyweights and food intakes were recorded during the maturation phase and physical development, learning ability, motor activity and reproductive performance were assessed. Details on measured offspring physical and functional development parameters are not reported but conformed to standard internationally accepted regulatory guidelines.

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2.5. Local tolerance study

3.2. Repeat dose toxicity study

In this study Polymite was injected subcutaneously, at a dose level of 1 ml per animal, into the left and right anterior sites of each of eight New Zealand White rabbits on day 1. A control formulation (1 ml of 20 mg/ml tyrosine blank + 0.05 mg/ml MPL® ), was similarly injected into the left middle side and 1 ml of physiological saline was injected into the right middle side of each rabbit. Skin from a central area, posterior to the treated sites, was identified as an untreated control. Groups of two males and two females were sacrificed on day 5 and the remaining animals were killed on day 10. Assessment of general health, systemic toxicity, and local reaction was made throughout the study; dose sites were evaluated macroscopically and histologically following the days 5 and 10 kills.

Single subcutaneous doses of Polymite at 0.1 and 1 ml per rat once weekly for 13 weeks were well tolerated and findings were restricted to the latter dose level. These findings comprised raised neutrophil count (and total white cell count) and raised spleen weight for both sexes. Values are shown in Table 3. None of these findings were seen after the 4-week recovery period. At the terminal kill, there were macroscopic (discoloration, fur loss, thickening and masses) and microscopic (cellulitis/fibrosis, abscess and haemorrhage) findings suggestive of local irritation at the injection site of both control and treated animals, however, the severity of the findings was generally greater in high dose animals. At the treatment-free recovery kill, there was evidence of partial recovery from both the macroscopic and microscopic observations. Other than the dose site changes, there were no study findings at 0.1 ml per rat.

3. Results

3.3. Reproduction toxicity studies

3.1. Single dose toxicity studies

In embryo-foetal studies, Polymite showed no maternal toxicity and no adverse effects on pregnancy rate, uterine/implantation data, foetal data or on foetal defect data in the rat or rabbit following subcutaneous administration at dose levels of 0.1 and 1 ml per animal on five occasions during gestation. Among rats, swelling in the neck region was observed occasionally in control and 0.1 ml Polymite dose groups during gestation and in the majority of 1 ml Polymite dose group animals. Necropsy examination revealed that this swelling corresponded to the presence of thickening/raised area/nodule or mass which was sometimes described as firm. In a pre- and post-natal development study, Polymite showed no maternal toxicity and no adverse effects on the F1 generation in the rat following subcutaneous administration at dose levels of 0.1 and 1 ml per animal on days 1, 6, 10, 14 and 17 of gestation. Swelling in the neck region was observed occasionally in the 0.1 ml Polymite dose group during gestation and lactation and in the majority of

There were no study findings for mice given a single subcutaneous dose of Polymite at dose volumes of 0.05 and 0.5 ml per mouse. The only findings in mice dosed by the intraperitoneal route were clinical signs of fine tremors and lethargy in controls, decreased activity, lethargy and pilo-erection with 0.05 ml Polymite and tremors, increased activity, vocalisation and lethargy with 0.5 ml of Polymite up to 1 or 2 h post-dose. Notable macroscopic changes were limited to cases of apparently enlarged livers in the two groups receiving intraperitoneal Polymite. For rats, there were no study findings following a single subcutaneous dose of Polymite at dose volumes of 0.1 and 1 ml per rat. The only finding of note following intraperitoneal administration was one male with an apparently enlarged liver (with a dark and mottled appearance) at the latter dose level.

Table 3 Effect of repeat subcutaneous administration of 1 ml per rat Polymite on various toxicity study parameters Parameter

Treatment

Value (change)

Control male 1 ml per animal male Control female 1 ml per animal female

1.5 4.4 (2.9-fold increase) 1.0 2.7 (2.7-fold increase)

Total white cell count (1000 cm2 )

Control male 1 ml per animal male Control female 1 ml per animal female

6.2 9.4 (1.5-fold increase) 4.3 6.2 (1.4-fold increase)

Spleen weight (g)

Control male 1 ml per animal male Control female 1 ml per animal female

0.716 0.804 (12% increase) 0.486 0.558 (15% increase)

Neutrophil count

(1000 cm2 )

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1 ml Polymite dose group animals. Necropsy examination, 3 weeks after the final dose administration, showed no findings. 3.4. Local tolerance study Single subcutaneous injections of 1 ml Polymite control (tyrosine blank + MPL® adjuvant) or physiological saline to the rabbit resulted in no dermal reactions or clinical signs. Macroscopic changes were limited to a pale raised area or red area at the Polymite and control injection sites (single males), marked redness at the control injection site (one male and one female) and slight redness at the saline of one female. Microscopic findings at Polymite injection sites revealed irritant responses, primarily foreign body granulomas, characterised by aggregates of macrophages, together with minor levels of polymorphs and necrotic debris. There were sometimes surrounding cystic spaces, and cellulitis/fibrosis as characterised by a mixed inflammatory cell infiltration of the subcutis, with varying levels of fibrosis. All the effects were in comparison with saline and untreated sites but were comparable with the degree of irritation seen at the control injection site. Findings at 10 days post-dosing were reduced in comparison with findings from 5 days after administration, suggesting partial recovery. 4. Discussion Published non-clinical data on the safety or efficacy of dust mite extracts are scant. A protective immune response assessed by immunoglobulin (Ig) response for polymerised D. pteronyssinus extract (using 0.5% glutaraldehyde) has been shown [35]. Guinea pigs were given a subcutaneous dose of two times 0.5 ml of 50% native extract or polymerised extract and 50% Freunds Complete Adjuvant. Results showed reduced reactivity with IgE antibody but similar or slightly enhanced immunogenicity as measured by IgG induction. The battery of non-clinical studies reported here to support safe clinical use of Polymite indicate a lack of toxicological findings. Single dose mouse and rat studies by the subcutaneous route showed no effects at levels greatly above a typical clinical dose volume of 1 ml for these type of materials. Thus, on a weight to weight basis, a safety margin of at least 850-fold (mouse) or 200-fold (rat) was achieved for a 60 kg person. Intraperitoneal administration resulted in some clinical signs of central nervous system (CNS) origin (including controls) which were considered to be largely attributable to the phenolic content (0.5% w/v) of the dose formulation. CNS toxicity has been reported in rodents for phenol [43]. The significance of apparently larger livers in some animals dosed by this route is not clear, although liver toxicity has been reported following repeated dosing of high levels of phenol in rodent studies [43].

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Repeated clinical doses of Polymite are envisaged and potential adverse effects were examined following 13 weekly doses to the rat. Findings of increased white cell parameters and spleen weight seen following once weekly administration of Polymite at 1 ml per rat (but not 0.1 ml per rat) are not unexpected considering that both tyrosine and MPL® are immunological adjuvants. The adjuvant properties of MPL® through immunostimulatory activity (producing diverse effects on the cellular elements of the immune system including macrophage activation and T and B cell interaction with concomitant cytokine and lymphokine release) are well covered in the literature [1,15,21,31,37]. The limited signs of immunostimulation seen following the 13 weeks treatment period were not seen following a 4-week non-dose period indicating a reversibility of the effects. Overall, this study showed that Polymite administered to the rat once weekly for 13 weeks elicited no toxicologically significant reactions. No systemic findings occurred at a dose level of 0.1 ml and not unexpected minor immunostimulatory effects were seen at a dose level of 1 ml. On the basis of a 1 ml clinical dose volume, these levels represent a safety margin of 20- and 200-fold, respectively, for a 60 kg person. Measurement for specific antibody response in the repeat dose toxicity study was not performed as it was considered unnecessary in the safety evaluation of Polymite. Also, although therapeutic allergy vaccines often induce the production of allergen specific IgG antibody, this is not now believed to be necessarily related to the efficacy of the products [8]. Thus, Polymite is designed to bring about much more subtle changes in the immune response at the T cell level, leading to a reduction of the inflammatory responses seen in allergic reactions. Reproduction toxicology studies in the form of embryofoetal (rat and rabbit) and pre- and post-natal (rat) investigations with Polymite showed that it had no reproductive effects following administration at 0.1 and 1 ml per animal on five occasions during gestation. As for the repeat dose toxicity study, these dose levels gave good safety margins (up to 200-fold) on the basis of a 1 ml clinical dose volume. A common response to any form of clinical vaccination is dose site reaction. A local tolerance study in the rabbit using a subcutaneous dose volume of 1 ml per animal Polymite showed minor macroscopic findings of infrequent reddening and notable microscopic findings of irritation of the dose site. These findings were similar to those seen with 1 ml per animal of tyrosine plus MPL® adjuvant. Evidence of some recovery within 10 days of dose administration was noted. In the repeat dose rat toxicity study, findings at the injection site of control (tyrosine formulation) and Polymite treated animals following once weekly administration were consistent with those of local irritation. Evidence of some recovery following a 4-week recovery period was noted. A local dose site reaction (notably swelling or nodules/masses) was seen in rats (including controls) in the reproduction toxicity studies.

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A potential for local irritation effects with Polymite is not unexpected for products of this nature. Aluminium salts (which have been used as adjuvants for many years in human vaccines) are known to produce local reactions in animals and clinically [11,18,32,38]. Local reactions have also been noted for other novel adjuvants under development [38]. The findings reported here are likely to be related to the presence of tyrosine in the dose formulation. As with the aluminium salts (often referred to as alum), it is believed that tyrosine acts as a short-term depot adjuvant [40]. A range of clinical studies using D. pteronyssinus tyrosine adsorbate have shown cases of local injection site reaction which has also occurred when a tyrosine alone formulation has been included as the placebo [2,9,17,20,28]. Further clinical studies using grass or tree tyrosine adsorbed pollen extracts have shown similar dose site reactions which have been mirrored when alum adsorbed extracts have been compared [3,22,34]. Thus, animal studies have shown a probable tyrosineinduced local irritation effect with Polymite, with evidence of reversibility. A further study in the Guinea pig has shown that tyrosine (up to 40 mg by subcutaneous injection) is removed gradually, but slowly and had cleared within 1 week, indicating the minimal possibility of permanent nodule formation [20]. However, it must be noted that there was evidence that the severity of the injection site findings in the rat (but not the rabbit) was greater when Polymite (notably at 1ml per rat) was compared to the tyrosine control, indicating that the rest of the Polymite formulation may have a role in this enhanced response. In conclusion, a specific preclinical safety testing strategy developed to support clinical use of a glutaraldehydemodified house dust mite allergy containing MPL® adjuvant (Polymite) and comprising single and repeat dose toxicity, reproduction toxicity and local tolerance studies, has shown that it produced no toxicological findings of significance at levels greatly in excess to those proposed for clinical use. A not unexpected, but relatively minor, immunostimulatory effect was seen following repeated dosing (once weekly for 13 weeks) at 1 ml per rat; the Polymite formulation also resulted in injection site reaction which can largely be attributed to the presence of tyrosine. No reproduction toxicity was found.

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