Local Tolerance Studies Research Articles

UPREGULATING ABCA1‐MEDIATED CHOLESTEROL TRANSPORTATION BY CS6253 – OPPORTUNITY FOR CONVENIENT SC ROUTE OF ADMINISTRATION

AbstractBackgroundThe ATP‐binding cassette transporter A1 (ABCA1) is a validated target for AD and upregulation has therapeutic potential in APOE4‐associated AD. The apoE4 protein compared to E2/E3 has impaired interaction with the ABCA1 transporter protein, affecting cholesterol homeostasis in the brain, causing dementia and hemorrhage. CS6253 is an ABCA1‐agonist that in monkey’s has shown safety and increase in Ab42/40‐ratio. A phase 1 SAD‐MAD with CS6253 administered IV was initiated. Here we describe translational efforts for transitioning from IV to SC route‐of‐administration.MethodThe Phase 1 is initiated with SAD:In 4‐6 cohorts of 8 subjects (6 active: 2 placebo) CS6253 starting with 1 mg/kg will be administered IV and plasma and CSF collected simultaneously over 24 hours to assess PK and response markers, and, MAD: In 3‐4 cohorts of 8 subjects (6 active: 2 placebo) CS6253 will be administered to elderly stratified for sex and APOE4 status.Using the identical CS6253 drug product, a GLP local‐tolerance study in rats was conducted to support up to 30 days SC administration in humans. Rat groups of n = 12 received PBS‐control and 7.5, 25 and 75 mg/kg CS6253 SC every other day, 7 times. PK is analyzed by LC‐MS following first and 7th/last dose.ResultBodyweight: Treated animals gained weight throughout the study at a comparable rate to control; no Test Article(TA)‐related variations identified.Clinical Signs: No TA‐related observations were noted, all animals remained in good condition until the end of dosing and end of recovery.Food consumption: No TA‐related effects on food consumption, all means were comparable to controls.Gross Pathology: No gross lesions were observed for terminal and recovery animals.Human Phase 1 SAD results are expected late summer of 2023 and human MAD data late fall of 2023 and with clearing local‐tolerance toxicology the CS6253 program can transition from IV to SC route‐of‐administration.ConclusionThe ABCA1 agonist CS6253 has therapeutic potential for APOE4‐associated AD. Safety, PK and biomarker efficacy is being assessed in a Phase 1 study. The SC local tolerance study opens up the potential to self‐administer CS6253 SC for optimal convenience and compliance.

Carbohydrate fatty acid monosulphate ester is a potent adjuvant for low-dose seasonal influenza vaccines

There is still a need for a better and affordable seasonal influenza vaccine and the use of an adjuvant could solve both issues. Therefore, immunogenicity of a combination of low dose of 1/5TH (3 µg of HA) a licensed seasonal flu vaccine with the novel carbohydrate fatty acid monosulfate ester (CMS)-based adjuvant was investigated in ferrets and safety in rabbits. Without CMS, hemagglutination inhibition (HI) antibody titers ranged from ≤5 to 26 three weeks post immunization 1 (PV-1) and from 7 to 134 post-immunization 2 (PV-2) in ferrets. Virus neutralizing (VN) antibody titers ranged from 20 to 37 PV-1 and from 21 to 148 PV-2. CMS caused 10 to 111- fold increase in HI titers and 3 to 58- fold increase in VN titers PV-1 and PV-2, depending on influenza strain and dose of adjuvant. Eight mg of CMS generated significantly higher antibody titers than 1 or 4 mg, while 1 and 4 mg induced similar responses.Three µg of HA plus 4 mg of CMS was considered the highest human dose and safety of two-fold this dose was determined in acute and repeated-dose toxicity studies in rabbits conducted according to OECD GLP guidelines. The test item did not elicit any clinical signs, local reactions, effect on body weight, effect on urine parameters, effect on blood biochemistry, or gross pathological changes. In blood, increased numbers of neutrophils, lymphocytes and/or monocytes were noted and in iliac lymph nodes, increased cellularity of macrophages of minimal to mild degree were observed. In both ferrets and rabbits, body temperature increased with increasing dose of CMS to a maximum of 1 ˚C during the first day post-immunization, which returned to normal values during the second day. In the local tolerance study, histopathology of the site of injection at 7 days PV-1 revealed minimal, mild or moderate inflammation in 5, 8 and 5 animals, respectively. In the repeated-dose study and 21 days PV-3, minimal, mild or moderate inflammation was observed in 15, 18 and 3 animals, respectively. We concluded that the data show CMS is a potent and safe adjuvant ready for further clinical development of a seasonal influenza vaccine and combines high immunogenicity with possible antigen-sparing capacity.

Effective wound healing agents based on N-alkenylimidazole zinc complexes derivatives: future prospects and opportunities

Introduction: The therapeutic effect of commercially available domestic and foreign drugs for the treatment of various skin injuries is far from optimal. These drugs have no universal effects, but cause pronounced side reactions. There is a clear demand for development of innovative wound-healing drugs with antimicrobial properties, which increase the natural protective function of the skin. Pharmaceutical compounds with zinc nanoparticles have been increasingly recognized as a promising therapeutic direction. These drugs can easily penetrate into damaged tissues and stimulate metabolic processes. Zinc complexes with imidazole derivatives are of a particular interest. Imidazole acts as a structural fragment of many natural physiologically active compounds, thus providing targeted delivery of this essential trace element into the wound for inclusion in the multicascade mechanism of wound healing. The aim of the study: to provide experimental evidence for effects of recently developed zinc complexes with N-alkenylimidazole as wound healing agents. Materials and Methods: Wound-healing effects of six 1% gels containing distinct N-alkenylimidazole zinc complexe derivatives based on the Na-carboxymethylcellulose (Na-CMC) were comparatively studied in 128 outbred white rats of both genders. The Na-CMC-based Zinc Sulfate 1% gel, Methyluracil and Solcoseryl served as reference drugs. After performing the local tolerance study of zinc complexes, linear and planar sterile wounds of comparable size were inflicted in anesthetized animals. The degree of healing was evaluated on the day 8 and day 28 after the treatment start by wound sizes and histological examination of inflammatory response, epithelization, granulation tissue, angiogenesis, and necrosis. The skin microcirculation system was evaluated using the laser Doppler Flowmetry (LDF), whereby the blood flow indicators were recorded 30 and 60 minutes after intraperitoneal administration of the trial compound. The antimicrobial activity of the zinc compounds was determined in vitro by means of their minimum inhibitory concentration suppressing the bacteria and fungi growth using the double serial dilution method in liquid culture media. The statistical data processing was performed using the Statistica 12 software package. Results and Discussion: In the linear wound model, all animals treated with either of six experimental zinc compounds showed almost complete reduction in wound size (92-100%, p<0.05) on the day 8, significantly exceeding the wound healing in the control animals (reduction by 67-88 %, p<0.05) and effects of the reference drugs (reduction by 83-86%, p<0.05). In the planar wound model, the most significant wound healing effect was reached by using the gel containing N-isopropenylimidazole zinc diacetate (encoded as Pilim-1). The respective histological examination showed signs of complete epithelialization, absence of destructive changes in the epidermis, restoration of skin appendages and presence of mature granulation tissue. Intraperitoneal Pilim-1 administration at a dose of 20 mg/kg improved microcirculation in the rat skin, as judged by significant effects on perfusion and the amplitudes of the isolated rhythms of the LDF-gram. In addition, Pilim-1 exerted a moderate bacteriostatic and fungistatic activity, which was 2 times greater than the antimicrobial activity of Metronidazole. Conclusion: Topical application of gels containing 1% N-alkenylimidazole zinc complex derivatives accelerates the healing of uninfected linear and planar wounds in comparison with the established reference drugs. The Pilim-1 zinc compound exhibited the most pronounced therapeutic effect. The observed in vitro antimicrobial action of Pilim-1 is of further interest for potential implications in treatment of infected skin wounds. The regenerative effect of this substance opens prospects for development of new drugs with improved wound healing properties.

Safety Study of an Original Mesenchymal Stromal Cell Secretome-Based Medicinal Product for Spermatogenesis Restoration

SCIENTIFIC RELEVANCE. Currently, there are no effective and safe medicinal products for idiopathic male infertility. Previous studies in two animal models of infertility (short-term cryptorchidism in rats and doxorubicin-induced testicular injury in mice) have shown the effectiveness of an originator medicinal product based on the mesenchymal stromal cell (MSC) secretome.AIM. The aim of the study was to evaluate the toxicity profile of the MSC secretome-based medicinal product in rats after local intratesticular or intramuscular administration.MATERIALS AND METHODS. The MSC secretome is a combination of factors secreted by MSCs in low-glucose Dulbecco’s modified Eagle’s medium (DMEM-LG) for MSC conditioning. In the single-dose toxicity study, the MSC secretome-based medicinal product was injected under the testicular tunica albuginea of male Wistar rats (15 per group) at doses of 15 and 25 relative units (RU) per animal, which are 1.5 and 2.5 times higher than the therapeutic dose (10 RU). In the repeat-dose toxicity study, male Wistar rats (10 per group) received intramuscular thigh injections of the medicinal product on days 1, 6, and 12 at doses of 15 and 25 RU per animal. The local tolerance study involved histopathological examination of the testes and thighs at the injection site. All studies included control groups of intact animals and animals similarly injected with blank DMEM-LG. The early follow-up period was 14 days, and the late follow-up period was 42 days.RESULTS. The rats showed no changes in the general condition after single and repeated doses of the MSC secretome-based medicinal product. Single subtunical doses induced moderate irritation; its signs included pathological changes in individual seminiferous tubules: epithelial atrophy (70% of the animals on day 14; 55% at late follow-up) and sperm stasis (70% of the animals). Similar changes were observed in the blank DMEM-LG group (up to 80% of the animals). There were no pathological changes in the tissues after repeated injections. A transient increase in alkaline phosphatase activity was detected in animals after their third intramuscular injection at a dose of 25 RU; the other biochemical parameters were normal in all study groups.CONCLUSIONS. The MSC secretome-based medicinal product has a favourable safety profile following both intratesticular and intramuscular administration, as it does not cause any permanent changes in the studied organs and tissues.

Evaluation of Developmental and Reproductive Toxicity in Rabbits for MB-102, a Fluorescent Tracer Agent Designed for Real-Time Measurement of Glomerular Filtration Rate.

The fluorescent tracer, MB-102, has been designed for the direct, real-time measurement of glomerular filtration rate. Previous studies, both in vitro and in vivo (rats, rabbits and dogs), were conducted to assess potential toxicity including single dose toxicity, mutation assay, chromosomal aberration assay, phototoxicity, local tolerance study, micronuclease assay, hERG channel changes, CNS and cardiovascular safety. The results of these studies led to a safety/toxicology profile for this agent deemed sufficient by the FDA to conduct Phase I and Phase II human clinical studies. In this paper we report on maternal toxicity and the potential effects on embryo-fetal development and the toxicokinetics of MB-102 administered daily via intravenous (bolus) injection into pregnant rabbits during the period of organogenesis gestation day 7-19. Assessment of toxicity was based on mortality, clinical observations, body weight, food consumption, reproductive performance and necropsy and cesarean section findings. Blood samples were collected for toxicokinetic evaluation. No test article findings were noted in any of these studies. The only clinical findings observed were the discoloration of skin, eyes or pelage in the 2 higher dose groups, which were considered related to the color and fluorescent properties of MB-102 and were deemed non-adverse. Exposure, as assessed by Cmax and AUC(0-6) increased in a dose dependent manner from 4.5 to 113mg/kg/day. No accumulation of the test article was noted after multiple doses were administered. Thus, intravenous administration of MB-102 was not associated with any adverse developmental or reproductive toxicities in pregnant rabbits.

RVSV-ΔG-SARS-CoV-2-S vaccine: repeated intramuscular (IM) toxicity, local tolerance, immunogenicity and biodistribution study in NZW rabbits.

BriLife®, a vector-based vaccine that utilizes the recombinant vesicular stomatitis virus (VSV) platform to express and present the spike antigen of SARS-CoV-2, is undergoing testing in a phase 2 clinical trial in Israel. A nonclinical repeated-dose (GLP) toxicity study in New Zealand white rabbits was performed to evaluate the potential toxicity, local tolerance, immunogenicity and biodistribution of the vaccine. rVSV-ΔG-SARS-CoV-2-S (or vehicle) was administered intramuscularly to two groups of animals (106, 107 PFU/animal, n = 10/sex/group) on three occasions, at 2-week intervals, followed by a 3-week recovery period. Systemic clinical signs, local reactions, body weight, body temperature, food consumption, ophthalmology, urinalysis, clinical pathology, C-reactive protein, viremia and antibody levels were monitored. Gross pathology was performed, followed by organs/tissues collection for biodistribution and histopathological evaluation. Treatment-related changes were restricted to multifocal minimal myofiber necrosis at the injection sites, and increased lymphocytic cellularity in the iliac and mesenteric lymph nodes and in the spleen. These changes were considered related to the inflammatory reaction elicited, and correlated with a trend for recovery. Detection of rVSV-ΔG-SARS-CoV-2-S vaccine RNA was noted in the regional iliac lymph node in animals assigned to the high-dose group, at both termination time points. A significant increase in binding and neutralizing antibody titers was observed following vaccination at both vaccine doses. In view of the findings, it was concluded that the rVSV-ΔG-SARS-CoV-2-S vaccine is safe. These results supported the initiation of clinical trials.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00204-022-03302-5.

Repeat-dose and local tolerance toxicity of SARS-CoV-2 FINLAY-FR-02 vaccine candidate in Sprague Dawley rats

This study evaluates safety of FINLAY-FR-02, a vaccine candidate against SARS-CoV-2 based on the recombinant receptor binding domain conjugated to tetanus toxoid, in a preclinical, repeat-dose toxicity and local tolerance study. Sprague Dawley rats were randomly allocated to three experimental groups: control (receiving physiological saline solution); placebo (receiving all vaccine components except antigens) and vaccine group (receiving three doses of the vaccine candidate, 37.5 µg of RBD) administered intramuscularly in hind limbs at 24 h intervals during three days. We evaluated physiological condition, pain, food and water consumption, body temperature, dermal irritability, injection site temperature and inflammation, immunological response, blood chemistry, relative organ weight, histopathology and immunotoxicology. The product was well tolerated; no clinically relevant changes, pain, local effects or adverse systemic toxicological changes or deaths were observed. These preliminary results permitted the Cuban regulatory authorities to authorize clinical trials in humans.

Saponin Micelles Lead to High Mucosal Permeation and In Vivo Efficacy of Solubilized Budesonide.

Due to fast nasal mucociliary clearance, only the dissolved drug content can effectively permeate the mucosa and be pharmaceutically active after intranasal application of suspensions. Therefore, the aim of this study was to increase the budesonide concentration in solution of a nasal spray formulation. Budesonide, a highly water-insoluble corticosteroid, was successfully solubilized using a micellar formulation comprising escin, propylene glycol and dexpanthenol in an aqueous buffered environment (“Budesolv”). A formulation based on this micellar system was well-tolerated in the nasal cavity as shown in a good laboratory practice (GLP) local tolerance study in rabbits. Ex vivo permeation studies into porcine nasal mucosa revealed a faster and more efficient absorption. Budesolv with 300 µg/mL solubilized budesonide resulted in a budesonide concentration of 42 µg/g tissue after only 15 min incubation. In comparison, incubation with the marketed product Rhinocort® aqua 64 (1.28 mg/mL budesonide as suspension) led to 15 µg/g tissue. The in vivo tumor-necrosis-factor (TNF)-α secretion in an acute lung inflammation mouse model was significantly reduced (p < 0.001) following a prophylactic treatment with Budesolv compared to Rhinocort® aqua 64. Successful treatment 15 min after the challenge was only possible with Budesolv (40% reduction of TNF-α, p = 0.0012) suggesting a faster onset of action. The data reveal that solubilization based on saponin micelles presents an opportunity for the development of products containing hardly soluble substances that result in a faster onset and a better topical treatment effect.

Acute toxicity “six-pack” studies supporting approved new drug applications in the U.S., 2015–2018

The acute toxicity “six-pack” is a battery of animal tests used to evaluate acute systemic toxicity by three routes of exposure, skin and eye irritation/corrosion, and skin sensitization. A perception exists that these tests are not required for pharmaceuticals. For the four years from 2015 through 2018, we tallied the number of corresponding tests submitted by sponsors in approved, original new drug applications, as reported by the U.S. Food and Drug Administration (FDA) in publicly available reviews. In 125 reviews, we identified 228 single dose acute toxicity studies, 62 in vivo local tolerance studies, and 32 in vivo skin sensitization studies, as well as 37 in vitro or ex vivo local tolerance studies. A total of 4798 animals were used in these studies; however, FDA's reporting was inconsistent, and we estimate the actual number of animals used to be 8998. For the evaluation of single dose acute toxicity, we accessed two guidance documents with conflicting recommendations regarding routes of administration and number of species to be used. For the evaluation of local tolerance and skin sensitization, most studies examined were conducted by routes other than that intended for human administration. Non-animal methods used to evaluate skin sensitization were not reported.

High-frequency (20MHz) high-intensity focused ultrasound system for dermal intervention: A 12-week local tolerance study in minipigs.

High-intensity focused ultrasound (HIFU) operating at 20MHz is new and applicable to skin. Details of use and instrumentation are not documented. A GLP compliant 12-week study of Göttingen minipigs (n=3) was undertaken. Effects of HIFU treatment at different focal depths, energy levels and field size (single shot vs 5×5 multiple shots) were studied. Clinical scoring and histology of treated sites were made. High-intensity focused ultrasound showed instant and initial effects with wheal and flare responses followed by delayed inflammatory reactions associated with outer skin necrosis, depending on energy dose. HIFU treatment was tunable in the range 0.3-1.5 J, ablative at higher energy level. Transducers with deeper focal points gave more profound effects, while epidermal effects were comparable. Multiple doses of 5×5 shots produced stronger reactions than single dose indicating that nearby applied shots were synergistic. Recovery from single doses was faster than in multidose areas. Clinical scarring at the end point was not seen despite occasional fibrous change of dermis. Findings illustrated intended therapeutic use; no special safety issues of concern were raised. The new 20MHz HIFU was reproducible, tunable and produced targeted effects in the outer skin, for example instant wheal and flare followed by inflammation and possibly necrosis depending on energy setting. Reactions recovered during the study with only minor findings at study end. No special safety concerns were raised. The method can be controlled and modulated, and it is ready for clinical testing of dermatological disease indications including conditions presently treated with lasers.

Safety Evaluation of PQ Birch Allergy Immunotherapy to Support Product Development

PQ Birch represents an allergen-specific immunotherapy for the treatment of birch pollinosis. It consists of native birch pollen extract chemically modified with glutaldehyde adsorbed to L-tyrosine in its microcrystalline form with addition of the adjuvant Monophosphoryl Lipid A (MPL®). A nonclinical safety testing strategy was designed based upon interpretation of current legislation and regulatory intelligence and comprised genotoxicity studies (bacterial reverse mutation and Chinese hamster ovary micronucleus assays), a rat repeat dose toxicology study and a rabbit local tolerance study. No safety findings of concern were found. Thus, no evidence of genotoxicity was found. Relatively minor, immunostimulatory effects were seen following repeated subcutaneous dosing (once every 2 weeks for 13 weeks) as reversible increased white cell count (notably neutrophils), increased globulin level (resulting in decreased albumin/globulin [A/G] ratio) and increased fibrinogen, as well as minor dose site reaction in the form of inflammatory cell infiltrate. These findings are likely due to the immunostimulatory nature of MPL® and/or the presence of L-tyrosine within the adjuvanted vaccine. Similar dose site inflammatory changes to the injected formulation were also noted in the rabbit local tolerance study.

New toxicity testing of PQ grass allergy immunotherapy to support product development.

PQ Grass represents an allergen-specific immunotherapy for pre-seasonal treatment of patients with seasonal allergic rhinitis (or rhinoconjunctivitis) with or without mild-to-moderate bronchial asthma. It consists of a native pollen extract for 13 grass species, chemically modified with glutaraldehyde, and adsorbed to l-tyrosine in a microcrystalline form with addition of the adjuvant Monophosphoryl Lipid A (MPL® ). Previous non-clinical safety testing, including rat repeat dose toxicity in adult and juvenile animals, rat reproductive toxicity and rabbit local tolerance studies showed no safety findings of concern. A new Good Laboratory Practice compliant rat subcutaneous repeat dose toxicity study to evaluate a higher clinical dose and modified posology (once every 2weeks for 13weeks) showed no signs of toxicity. As seen in previous studies, relatively minor, immunostimulatory effects were seen such as reversible increased white cell count (notably neutrophils), increased globulin level (resulting in decreased A/G ratio) and increased fibrinogen as well as minor dose site reaction in the form of inflammatory cell infiltrate. These findings are likely due to the immunostimulatory nature of MPL and/or the presence of l-tyrosine within the adjuvanted vaccine. This new toxicity study with PQ Grass therefore supports longer posology with higher dose levels.

Development of a nasal spray containing xylometazoline hydrochloride and iota-carrageenan for the symptomatic relief of nasal congestion caused by rhinitis and sinusitis.

IntroductionXylometazoline hydrochloride (HCl) is a nasal decongestant that causes vasoconstriction in the nasal submucosa. It has been used for more than 50 years for the treatment of nasal congestion caused by rhinitis/sinusitis. Iota-carrageenan is effective against a broad variety of respiratory viruses, which are the most common cause of infections of the upper respiratory tract. Therefore, it is used as the active component in the antiviral nasal spray Coldamaris prophylactic (1.2 mg/mL iota-carrageenan in 0.5% NaCl) and other medical device nasal sprays that are approved and marketed in the EU. Recently, we developed a nasal spray formulation containing both xylometazoline HCl (0.05%) and iota-carrageenan (0.12%) that provides decongestion and antiviral protection of the nasal mucosa at the same time.ResultsA set of in vitro experiments revealed that the vasoconstrictive properties of xylometazoline HCl and the antiviral effectiveness of iota-carrageenan against human rhinovirus (hRV) 1a, hRV8 and human coronavirus OC43 were maintained in the formulation containing these two compounds. Permeation experiments using bovine nasal mucosa showed that iota-carrageenan had no significant influence on the permeation of xylometazoline HCl. Finally, in the local tolerance and toxicity study, it was shown that the formulation was well tolerated at the application site with no occurrence of erythema or edema in the nostrils of all rabbits or any signs of toxicity in any of the organs and tissues inspected.ConclusionInvestigations on compatibility of xylometazoline HCl and iota-carrageenan demonstrated that the substances do not influence each other, allowing both to fulfill their known specific clinical efficacy (xylometazoline HCl) and effectiveness (iota-carrageenan).

Current progress of research and use of microminipigs in drug development

The use of minipigs has been increasing in the areas of pharmacology researches and drug development. The microminipig developed by Fuji Micra Inc. (Shizuoka, Japan) inherits characteristics of other pig strains showing several similarities to humans in anatomy, physiology, omnivorousness and diurnal, but at the same time has several advantages over other pig strains because of its small size which allows easy keeping, handling and dosing, and saving of test substances. The microminipig weighs about 10 kg at the age of 6 months. Canine cages can be used to keep the animal. Swine leukocyte antigens (SLA) are defined in each individual animal which is useful for testing immunological reactions. As there are many similarities in metabolic enzymes and transporters to those in humans, the microminipig is a powerful animal model for toxicokinetic studies. Unfortunately as in other minipigs the microminipig is not appropriate for embryo-fetal development studies of antibody drugs due to its poor placental transfer, but can be used for other reproductive and developmental studies. Repeat dose toxicity, safety pharmacology, immunotoxicity and local tolerance studies should be also other arenas of this animal model.

Towards Development of a Dermal Pain Model: In Vitro Activation of Rat and Human Transient Receptor PotentialAnkyrin Repeat 1 and Safe Dermal Injection of o-Chlorobenzylidene Malononitrile to Rat.

During clinical development of analgesics, it is important to have access to pharmacologically specific human pain models. o-Chlorobenzylidene malononitrile (CS) is a selective and potent agonist of the transient receptor potential ankyrin repeat 1 (TRPA1), which is a transducer molecule in nociceptors sensing reactive chemical species. While CS has been subject to extensive toxicological investigations in animals and human beings, its effects on intradermal or subcutaneous injection have not previously been reported. We have investigated the potential of CS to be used as an agonist on TRPA1 in human experimental pain studies. A calcium influx assay was used to confirm the capacity of CS to activate TRPA1 with >100,000 times the selectivity over the transient receptor potential vanilloid receptor 1. CS dose-dependently (EC50 0.9μM) released calcitonin gene-related peptide in rat dorsal root ganglion cultures, supporting involvement in pain signalling. In a local tolerance study, injection of a single intradermal dose of 20mM CS to rats resulted in superficial, circular crusts at the injection sites after approximately 4days. The histopathology evaluation revealed a mild, acute inflammatory reaction in the epidermis and dermis at the intradermal CS injection site 1day after administration. After 14days, the epidermal epithelium was fully restored. The symptoms were not considered to be adverse, and it is suggested that doses up to 20μL of 20mM CS can be safely administered to human beings. In conclusion, our data support development of a CS human dermal pain model.

Non-clinical safety evaluation of intranasal iota-carrageenan.

Carrageenan has been widely used as food additive for decades and therefore, an extended oral data set is available in the public domain. Less data are available for other routes of administration, especially intranasal administration. The current publication describes the non-clinical safety and toxicity of native (non-degraded) iota-carrageenan when applied intranasally or via inhalation. Intranasally applied iota-carrageenan is a topically applied, locally acting compound with no need of systemic bioavailability for the drug’s action. Animal experiments included repeated dose local tolerance and toxicity studies with intranasally applied 0.12% iota-carrageenan for 7 or 28 days in New Zealand White rabbits and nebulized 0.12% iota-carrageenan administered to F344 rats for 7 days. Permeation studies revealed no penetration of iota-carrageenan across nasal mucosa, demonstrating that iota-carrageenan does not reach the blood stream. Consistent with this, no relevant toxic or secondary pharmacological effects due to systemic exposure were observed in the rabbit or rat repeated dose toxicity studies. Data do not provide any evidence for local intolerance or toxicity, when carrageenan is applied intranasally or by inhalation. No signs for immunogenicity or immunotoxicity have been observed in the in vivo studies. This is substantiated by in vitro assays showing no stimulation of a panel of pro-inflammatory cytokines by iota-carrageenan. In conclusion, 0.12% iota-carrageenan is safe for clinical use via intranasal application.

Evaluation of the toxicology, pharmacokinetics, and local tolerance of recombinant factor IX Fc fusion protein in animals

IntroductionRecombinant factor IX Fc fusion protein (rFIXFc) is a recombinant coagulation factor composed of a single molecule of recombinant factor IX (rFIX) covalently fused to the Fc domain of human immunoglobulin G1 (IgG1) with no intervening sequence. An extensive nonclinical program was performed to support the clinical development of rFIXFc for treatment of people with hemophilia B. Materials and MethodsRepeat-dose toxicology studies of rFIXFc were performed in 2 relevant species: Sprague Dawley rats (4-week study) and cynomolgus monkeys (5- and 27-week studies). Assessments included in-life observations, electrocardiograms (monkeys only), laboratory evaluations (including hematology and blood chemistry), postmortem analyses, local tolerance, and pharmacokinetics (PK). Allometric scaling was performed with PK data from multiple species, including humans. Local tolerance (single-dose study) and thrombogenic potential (Wessler stasis model) of rFIXFc were tested in New Zealand White rabbits. ResultsThere were no significant local or systemic toxicity findings in the repeat-dose studies. Allometric scaling data suggested that animal rFIXFc PK results are predictive of human PK parameters. There were no findings from the local tolerance study in rabbits; thrombogenic activity was less than that elicited by rFIX and a prothrombin complex concentrate, and similar to vehicle control. ConclusionsrFIXFc was well tolerated in toxicology studies and demonstrated a low thrombogenic potential. These results are consistent with phase 1/2a and phase 3 clinical studies of rFIXFc in people with hemophilia B.

Preclinical evaluation of a Haemophilus influenzae type b conjugate vaccine process intended for technology transfer

Introduction of Haemophilus influenzae type b (Hib) vaccine in low- and middle-income countries has been limited by cost and availability of Hib conjugate vaccines for a long time. It was previously recognized by the Institute for Translational Vaccinology (Intravacc, originating from the former Vaccinology Unit of the National Institute of Public Health [RIVM] and the Netherlands Vaccine Institute [NVI]) that local production of a Hib conjugate vaccine would increase the affordability and sustainability of the vaccine and thereby help to speed up Hib introduction in these countries. A new affordable and a non-infringing production process for a Hib conjugate vaccine was developed, including relevant quality control tests, and the technology was transferred to a number of vaccine manufacturers in India, Indonesia, and China. As part of the Hib technology transfer project managed by Intravacc, a preclinical toxicity study was conducted in the Netherlands to test the safety and immunogenicity of this new Hib conjugate vaccine. The data generated by this study were used by the technology transfer partners to accelerate the clinical development of the new Hib conjugate vaccine.A repeated dose toxicity and local tolerance study in rats was performed to assess the reactogenicity and immunogenicity of a new Hib conjugate vaccine compared to a licensed vaccine. The results showed that the vaccine was well tolerated and immunogenic in rats, no major differences in both safety and immunogenicity in rats were found between the vaccine produced according to the production process developed by Intravacc and the licensed one. Rats may be useful to verify the immunogenicity of Hib conjugate vaccines and for preclinical evaluation.In general, nonclinical evaluation of the new Hib conjugate vaccine, including this proof of concept (safety and immunogenicity study in rats), made it possible for technology transfer partners, having implemented the original process with no changes in the manufacturing process and vaccine formulation, to start directly with phase 1 clinical trials.

Preclinical Safety of Baxter's Recombinant ADAMTS13

Abstract 3381Baxter is currently developing a recombinant ADAMTS13 (rADAMTS13, a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13) product for treatment, and routine prophylaxis of acute episodes of thrombotic thrombocytopenic purpura (TTP) in patients with ADAMTS13 deficiency. TTP is a rare, life-threatening microvascular disease characterized by single or recurrent episodes of thrombocytopenia, microangiopathic haemolytic anaemia and widespread microvascular thrombosis, which leads to the ischemic damage of multiple organs.To characterize the safety and the no observed adverse effect level (NOAEL) of Baxter's rADAMTS13, a 1-month repeat dose toxicity study in rats, an escalating dose and pilot 4-week repeat dose toxicity study in cynomolgus monkeys, and a 1-month repeat dose toxicity study including cardiovascular/pulmonary safety pharmacology in cynomolgus monkeys was conducted. Species suitability was shown by demonstrating that human rADAMTS13 can cleave monkey and rat VWF in vitro and in vivo under native conditions to present a worst case scenario for repeat dose toxicity studies. A local tolerance study was conducted in rabbits.Baxter's rADAMTS13 was well tolerated in rats at doses of 80, 400 and 800 FRETS-U/kg administered every third day for one month. Thus, the NOAEL in rats was the highest dose of 800 FRETS-U/kg. In cynomolgus monkeys, no adverse effects were detected at 800 FRETS-U/kg after intravenous treatment with rADAMTS13 once a week for 5 weeks (pilot repeat dose study). The NOAEL for the escalating dose phase of the study was 1790 FRETS-U/kg. Treatment of cynomolgus monkeys with bolus injection of Baxter's rADAMTS13 at doses of 80, 200, and 400 FRETS-U/kg once a week for 29 days did not reveal adverse findings. Thus, the NOAEL for this study was the highest dose of 400 FRETS-U/kg. As expected for a heterologous human protein drug, repeat doses of rADAMTS13 resulted in the formation of anti-drug antibodies specific for human ADAMTS13 and in neutralising human ADAMTS13 activity in animal models. No injection site reactions were observed in animals of either species. rADAMTS13 was well tolerated after intravenous (intended clinical administration route), intraarterial and paravenous administration in rabbits.In conclusion, Baxter's rADAMTS13 was well tolerated in rats, monkeys and rabbits, with no toxicity observed even at the highest dose levels. Disclosures:Piskernik:Baxter Innovations GmbH: Employment. Dietrich:Baxter Innovations GmbH: Employment. Kubik:Baxter Innovations GmbH: Employment. Ruthsatz:Baxter Innovations GmbH: Employment. Scheiflinger:Baxter Innovations GmbH: Employment. Schwarz:Baxter Innovations GmbH: Employment. Muchitsch:Baxter Innovations GmbH: Employment.

Study designs for the nonclinical safety testing of new vaccine products

During the development of a new vaccine, the purpose of nonclinical studies is to provide safety information to support the clinical development and licensure of the product. In this article the study designs currently accepted for the nonclinical safety testing of new vaccines are described for single dose, local tolerance, repeat dose toxicity and safety pharmacology studies; these studies together form the basis of a typical nonclinical safety evaluation dossier. The detailed design of the preclinical package must take account of the intended clinical use, patient population, route of administration, formulation, dose level and immunisation schedule. The test item that is used for these studies must be adequately representative of the intended clinical formulation. The animal model used for these studies must be selected on criteria of relevance. Single dose toxicity studies provide information on acute actions or the potential effect of accidental overdose, but this information is often available from the repeat dose toxicity study, obviating the need for the acute study. Local tolerance studies provide information on tissue reactions at the site of administration. Evaluation of the findings must distinguish between normal tissue responses to injected material and findings indicative of undesirable pathological changes. The repeated dose toxicity studies are the principal studies that support the safety profile of the vaccines. The design of these studies must take full account of the features of the vaccine in the choice of treatment regime, dose levels, pharmacodynamic monitoring and timing of investigations and sacrifice. Safety pharmacology studies are performed to evaluate the potential for undesirable secondary pharmacological actions of vaccines if there is data to suggest that such studies are needed; this evaluation is made on a case by case basis. In the absence of specific guidance the design of studies for therapeutic vaccines follows the same general principles as those for anti-infective vaccines.