Selected Biological Medicinal Products and Their Veterinary Use

Simple Summary Biological drugs are a generation of drugs that have developed thanks to advances in genetic engineering and molecular biology. Biological drugs are proteins derived from living cells or obtained through the use of genetic engineering methods with a selective and specific mechanism of action. Currently, these drugs are widely used in the treatment of many human diseases, but an increasing number of drugs from this group are also being used in the treatment of animals, mainly in dermatology, rheumatology and oncology. Abstract Definitions of biological medicinal products (BMPs) vary depending on the source. BMPs are manufactured using complex biological/biotechnological processes involving living cell lines, tissues and organisms such as microorganisms, plants, humans and even animals. Advances in modern biotechnological methods and genetic engineering have made it possible to search for new drugs with a targeted effect and simultaneous reduction of adverse effects, which has resulted in BMPs dynamically increasing their share in the pharmaceutical market. Currently, these drugs are widely used in the treatment of many human diseases, but an increasing number of drugs of this group are also being used in the treatment of animals, mainly in dermatology, rheumatology and oncology. This article presents the current state of knowledge in the field of biological medicinal products used in animal therapy.


Introduction
Biological medicinal products (BMPs) are a group of medicines which are developing rapidly owing to the progress of biotechnological methods. In terms of structure, these are proteins with pharmacological activity, derived from living cells or obtained by genetic engineering methods. The action of BMPs involves mimicking the function of normal animal proteins. Moreover, they act as modulators of immune response, because they mobilize the immune potential of the patient in the fight against the disease [1]. Biological drugs include the following groups: vaccines, blood proteins, toxins, recombinant proteins, monoclonal antibodies, growth hormone, insulins, erythropoietin, interferons, growth factors and interleukins. BMPs are used in the treatment or prevention of cardiological, dermatological, rheumatological and oncological diseases, Turner's syndrome, diabetes, anemia, oncological and neutropenia [2]. According to the United States Department of Agriculture biosimilar medicine, defined as a biological product that is FDA approved because it is similar to an FDA approved biological reference product and has been shown to have no clinical differences from this product [17]. The first biosimilar drug approved by EMA and placed on the European market in 2006 was the human recombinant growth hormone in the Omnitrope (Sandoz GmbH, Langkampfen, Austria) product, which is a generic drug for Genotropin. The second level, however, is interchangeable biosimilar products, defined as a biosimilar product to an FDA approved reference medicine and it is possible that they have the same clinical effect as the reference medicine and can therefore be used interchangeably [20]. However, no interchangeable biosimilar products have been approved.

Classification of Biological Medicinal Products
There are several groups of BMPs: monoclonal antibodies, stem cells, fusion (chimeric) proteins, recombinant proteins and antisense oligonucleotides [1,21].

Monoclonal Antibodies
Monoclonal antibodies are immunoglobulins, which specifically bind to proteins located on the surface of cells and contribute to the immune response. They act as immunosuppressants or immunostimulants [22]. The first monoclonal antibodies were of murine origin; however, when introduced to the human body, they produced an immune response against the foreign-species murine immunoglobin (HAMA-human anti-mouse antibody response). For this reason, attempts at humanization of antibodies were made to reduce their immunogenicity [1]. They resulted in obtaining chimeric antibodies, which were in 65-90% human antibodies (the percentages relate to amino acid sequence conservation), by replacing a fixed region of the heavy and light chain of the murine antibody with similar fragments of the human antibody-that is, only the replaced regions are of murine origin. Subsequently, humanized antibodies were produced which were 90-95% human; they were obtained by leaving only the antigen-binding regions in the murine antibody molecule. Currently, fully human antibodies are available, produced by transgenic mice, in which loci of murine DNA were replaced with DNA encoding human immunoglobulins [23]. The names of monoclonal antibodies end with -ab; apart from that, the name ending identifies the antibody type: chimeric antibodies, -ximab; humanized antibodies, -zumab; human antibodies, -umab [1,21].
The monoclonal antibody (mAb) timing system has been developed, resulting in an international non-proprietary name (INN) introduced at the request of the World Health Organization (WHO) or name adoption (USAN) at the request of the American Medical Association (AMA) in the United States [24][25][26]. According to these guidelines, the name mAb consists of the prefix chosen, the target sub-element and the end of the universal core "-mab". For monoclonal antibodies used in veterinary medicine, the target element is 'vet' [27].

Stem Cells
Mesenchymal stem cells (MSCs) are multipotent cells, with capacity for self-renewal and differentiation that derive from the embryonic layer of the mesoderm and under certain conditions they can differentiate into can myocytes, neural cells, osteoblasts, chondrocytes [28]. In animals, two classes of stem cells have been identified: embryonic stem cells (ESC) and adult stem cells (ASC) [29], which include hematopoietic stem cells, mesenchymal stem cells and progenitor cells [30]. In veterinary medicine, MSCs are used for treatment of tendon and ligament injuries, and joint diseases, with significant clinical relevance in horses and for orthopedic applications in dogs. Most often, MSCs are isolated from: adipose tissue, peripheral blood, umbilical cord, muscle, bone marrow, synovium [31]. The low immunogenicity of these cells suggests that MSCs can be transplanted universally without matching between donors and recipients. The transplantation possibilities for peripheral blood MSCs include allogeneic stem cells being injected from another donor animal from the some species, autologous stem cell being injected within the same animal and xenogeneic stem cell being injected from another donor animal from another species [32].

Fusion Proteins
The molecules of fusion proteins consist of two elements which make up a structure resembling an antibody. The first part has two binding domains which recognize a specific receptor protein. The other part is the Fc fragment of human immunoglobulin and it stabilizes the whole structure. The immunogenicity of these proteins is very low [22]. The names of fusion proteins end with -cept [1,33]. However, fusion proteins are not used in veterinary medicine.

Recombinant Proteins
Recombinant proteins are replicas of natural proteins or their fragments. They act specifically through cell receptors, inducing a precise immune response. This group includes interferons, interleukins, growth factors and protein hormones [22]. The names of recombinant proteins start with prefix rhu- [1,34].

Antisense Oligonucleotide
The antisense oligonucleotide group contains few drugs. These are short synthetic molecules, 12-30 nucleotides long, whose mechanism of action involves inhibition of gene expression as a result of blocking the relevant matrix RNA (mRNA) by an external complementary fragment of RNA, which binds selectively with mRNA of the gene of interest, blocks it and, in effect, prevents production of the relevant protein [35]. These drugs are indicated in selected neurodegenerative diseases [36], viral infections [37], cancers [38] and familial hypercholesterolemia [39].

BMPs Approved by EMA for Veterinary Use
BMPs approved by EMA (European Medicines Agency) for veterinary use are listed in Table 1. Virbagen Omega (Virbac, Carros, France) is available as lyophilizate with a solvent, from which suspension for injection is prepared. Effectiveness: a reduction in mortality of 4.4-6.4 times was observed in the treatment of dogs with enteric form of parvovirosis. In the treatment of cats infected with feline leukaemia virus (FeLV), a reduction in clinical symptoms was observed over four months and a reduction in mortality. In cats infected with feline immunodeficiency virus (FIV), mortality was low and did not change as a result of treatment [40] The effectiveness of the preparation in dogs and cats has been confirmed by many authors [41][42][43]. Dogs with parvoviral enteritis given intravenous omega interferon showed a significant improvement in clinical symptoms and a reduction in mortality by 4.4 times compared to control animals [41]. Cats with clinical signs related to feline leukemia virus (FeLV) infection and FeLV co-infection/feline immunodeficiency virus (FIV) receiving subcutaneous recombinant interferon were shown to have significantly lower mortality rates compared to the control group (39% versus 59% in 9 months and 47% compared to 59% in 12 months after therapy) [42]. In dogs receiving Virbagen omega subcutaneously, mean Canine Atopic Dermatitis Extent and Severity Index (CADESI) values improved from 42 on day 0 to 27 on day 120, overall by 36%. Pruritus scores decreased from 5.5 to 4.2, overall by 24%. Mean treatment outcomes slightly increased from 18 to 20. Overall outcomes improved from a mean of 65 to 52, overall by 21% [43]. After preparation, the solution is injected to sponges made of type II bovine collagen and grafted directly at the site of a long bone fracture. Subcutaneous injection at five places around the tumor removal site: one injection at each corner and one at the center of the square formed in the middle of the post-surgery scar, at approx. 0.2 mL per injection. The treatment regime is based on four injections every week (days 0, 7, 14, and 21), followed by two injections given every two weeks (days 35 and 49). The treatment should start one day earlier before the beginning of radiotherapy [48,49] Pegbovigrastim/Imrestor/ recombinant proteins Germany, 2015 The product increases the number of circulating neutrophils. It has also been proved that it enhances myeloperoxidase hydrogen peroxide halide mediated microbiocidal capabilities of neutrophils.

IU/vial Cattle (dairy cows and heifers)
Reduce the risk of clinical mastitis in periparturient dairy cows and heifers.
subcutaneous injection, one pre-filled syringe seven days prior to the expected calving and another 24 h thereafter. Injection of the contents of one pre-filled syringe ensures the administration of pegbovigrastim at 20-40 µg/kg b.w. TruScient, (Zoetis, Louvain-la-Neuve, Belgium) available as lyophilizate and a solvent for the preparation of a solution, was another product placed on the market. Effectiveness: simultaneous treatment of dogs with TruScient together with standard surgical care shortened the time needed for healing fractures. After 18 weeks, the fractures of all the dogs treated with TruScient together with standard surgical care (84) were seen to have healed, compared with 95% of the dogs treated with standard surgical care alone (40 out of 42). The product must not be used in dogs with a known hypersensitivity to the active substance or to any of the excipients or in dogs that are skeletally immature, have an active infection at the operative site, pathological fracture, or any active malignancy [44]. There is no information in the available literature describing the effects of recombinant human bone morphogenetic protein-2 for the treatment of open bone fractures in animals. A human study showed that 1.50 mg/mL of this substance was well above the standard of care in reducing the incidence of secondary interventions and general invasive procedures, accelerating fractures and wound healing, and reducing the rate of infections in patients with open fractures tibia [45]. TruScient was withdrawn from the European Union market at a request of the marketing authorization holder.
Subsequently, ProZinc (Boehringer Ingelheim Vetmedica GmbH, Duluth, GA, USA) was introduced to the market; it is in the form of a suspension for injection. Effectiveness: in a field study involving diabetic cats of different ages and breeds, after 6 weeks of treatment with ProZinc average blood sugar levels decreased and clinical symptoms improved. In an EU field study involving diabetic dogs, ProZinc was as effective as approved veterinary insulin. There has been an improvement in the measurement results of at least one blood sugar indicator and amelioration of at least one of three clinical symptoms: body weight, polyuria and polydipsia. The product must not be used in animals with hypersensitivity to the active substance or in emergencies associated with diabetic ketoacidosis [46]. It has been shown that in diabetic cats treated with protamine-zinc insulin, the probability of remission was 56.2%, and the median survival time (time from diagnosis to death) was 1488 days [47], which confirms the effectiveness of this drug.
Oncept IL-2 (Merial, Lyon, France) was introduced to the market in the same year (2013); it is available as lyophilizate and a solvent for the preparation of a suspension for injection. Effectiveness: the studies showed that in cats treated with Oncept IL-2, tumors returned longer (over 730 days based on mean range value) compared to control cats (287 days). Oncept IL-2 reduced the risk of relapse six months after starting treatment by approximately 56% after a year and 65% after two years [48]. It was shown that the recurrence of idiopathic fibrosarcomas was observed in 28% of cats receiving recombinant human interleukin-2 in the Canarypox virus compared to 61% of control animals during the 12-month follow-up after surgery [49].
Imrestor (Elanco GmbH, Cuxhaven, Germany) is another product available on the market; it is in the form of a solution for injections. Effectiveness: in a field study involving 2465 cows the incidence of clinical mastitis during days 3 to 30 of milk production was 9.1% (113/1235) in the Imrestor group compared with 12.4% (152/1230) in the group receiving indifferent injection. The relative reduction in incidence of mastitis was 26%. The product is contraindicated in cows with hypersensitivity to the active substance [50]. It was observed that animals treated with pegbovigrastim showed a 4-to 5-fold increase in circulating neutrophils within 24 h of starting treatment. Animals receiving pegbovigrastim showed a 35% reduction in the incidence of clinical mastitis compared to the control group during the first 30 days of lactation [51].
Subsequently, the product Cytopoint (Zoetis, Louvain-la-Neuve, Belgium) was introduced to the market, which is available as a solution for injections. Effectiveness: in a field study involving dogs with atopic dermatitis, 142 dogs received Cytopoint every month for 3 months, and 132 dogs were treated with ciclosporin (a drug approved for the treatment of atopic dermatitis). Cytopoint was as effective as ciclosporin in treating itchy skin. After 28 days, the pruritus score decreased by 52% in dogs receiving Cytopoint and 44% in dogs receiving cyclosporin. The product is contraindicated in dogs with hypersensitivity to the active substance and in those with a body weight under 3 kg [52]. A 51.9% reduction in pruritus was demonstrated in dogs treated with lokivetmab compared to 43.72% in dogs treated with cyclosporine after 28 days of dosing [53]. This randomized clinical trial confirms the effectiveness of lokivetmab in dogs with atopic dermatitis.
Arti-Cell Forte (Global Stem cell Technology (GST) NV, Evergem, Belgium) was introduced to the market one year later; it has the form of a suspension for injection. Effectiveness: in a field study involving horses with a history of lameness lasting 2 to 6 months, 50 horses were treated with Arti-Cell Forte and 25 received a placebo (dummy injection) of saline in one fetlock joint. All horses received intravenous ketoprofen, a medicine to relieve pain and inflammation, at the time of treatment. Six weeks after treatment 68% of the horses treated with Arti-Cell Forte had a lameness score reduction by 2 or 3 lameness grades compared to none of the horses in the control group. The product must not be used in animals with hypersensitivity to the active substance [54]. Significant improvement in lameness was observed in horses receiving intra-articular injections with chondrogen-induced mesenchymal stem cells and equine allogeneic plasma compared to the saline control group [55] which confirms the efficacy of this drug in the treatment of osteoarthritis.
The latest product that has obtained the marketing authorization is HorStem (Industrial Ventorro del Cano, Madrid, Spain). It is a suspension for injection. Effectiveness: in a field study of horses with mild to moderate osteoarthritis, 16 horses received an injection of HorStem into the affected joint and 17 horses received a placebo (dummy) injection. Horses were examined at days 14, 35 and 63. Twelve HorStem-treated horses were successfully treated (success rate 75%) at day 63 compared to only four of the control group (success rate 25%). A contraindication to the use of the preparation is hypersensitivity to the active substance [56]. The effectiveness of the equine umbilical cord mesenchymal stem cells was described in previous studies, which demonstrated that 77% of horses receiving these cells returned to work at the same or higher levels [57].

BMPs Approved by USDA for Veterinary Use
According to the USDA there are nine groups of BMPs and the last one (miscellaneous) is characterized in this subsection and in Table 2.
Eqstim (Neogen Corporation, Lexington, KY, USA) is available as a suspension for injection [58]. In a study carried out on horses that received Propionibacterium acnes, the immunostimulatory and immunomodulatory properties were characterized by increased expression of CD4 + T cells and lymphokine activated killing (LAK) activity in peripheral blood and bronchoalveolar fluid, increased nonopsonized phagocytosis in peripheral blood leukocytes and decreased pulmonary cellularity [59].
Vetimmune (Sass & Sass, Inc, Oak Ridge, TN, USA) is available as an oral solution [60]. The effectiveness of a polyprenyl immunostimulant was confirmed (in the double-blinded, randomized, placebo-controlled clinical trials in cats experimentally infected with feline herpesvirus type 1 [61]. Oncept (Merial, Lyon, France) is available as a vaccine [62]. The active substance is xenogenic plasmid DNA containing canine DNA of human tyrosinase (huTyr). The efficacy of a vaccine containing plasmid DNA with an insert encoding human tyrosinase confirms data published by Grosenbaugh et al. [63] in which the survival time of dogs suffering from oral malignant melanoma was significantly improved after treatment with the huTyr vaccine.
Zelnate (Diamond Animal Health, Inc., Des Moines, IA, USA) is available as a lyophilisate with two solvents, from which a solution for injection is prepared. The active substance of the preparation is produced by bacterial plasmid DNA with a liposomal carrier [64]. However, the new available data indicate that Zelnate does not appear to be an effective in the metaphylactic therapy of the bovine respiratory disease [65].
Victrio (Bayer AG, Leverkusen, Germany) is available as a lyophilisate with two solvents that are made into a suspension for injection. The active substance of the product is produced by bacterial plasmid DNA with a liposome carrier [66]. It is demonstrated that Victrio specifically and potently activates recombinant chicken toll-like receptor 21 (TLR21) in a nuclear factor kappa B reporter gene assay [67].    Immunotherapy of sarcoid tumors in horses.
1 mL of product per cubic centimeter of tumor; treatment should be repeated every 1 to 3 weeks until the tumor is resolved (generally repeated at 10-to 14-day intervals). [70,71] * IFN-gamma-Interferon-gamma.
Staphage Lysate (Delmond Laboratories, Swarthmore, PA, USA) is available as a vaccine [68]. In dogs with canine atopic dermatitis (CAD) and recurrent bacterial pyoderma that received Staphylococcus aureus Phage Lysate Staphage Lysate an effectiveness of 83.33% for the control of pruritus along with regression of the lesions was observed which confirms the efficiency of such a treatment [69].
Immunocidin Equine (NovaVive Inc., Greater Napanee, ON, Canada) is available as a suspension for injection [70]. A study in horses with sarcoids that received Immunocidin Equine showed that out of 17 cases, nine cases were completely healed (52.9%), three had improved (tumors shrank) (17.6%), and three cases had weight loss, tumors grew or did not resolve (17.6%), and in one case with two tumors, one tumor resolved and the other tumor had a slight regrowth [71].

Hormone Drugs
As this listing shows, the number of BMPs (especially recombinant drugs) approved by EMA and USDA in recent years for animal use is relatively small. This is a consequence of several facts, one of them being that the cost of introducing an original drug to the market is very high; therefore, the target group of patients (animals) should be large enough to ensure a sufficiently large market with a high drug price. Another reason could be the ease of obtaining effective active substances. Hormonal drugs of the gonadotropin group could be a good example. Currently recombinant follitropin alfa (Gonal-F, Merck Serono S.p.A., Modugno, Italy; Luveris, Merck Serono S.p.A., Modugno, Italy) and beta (Follistim, Merck Sharp & Dohme Corp., Whitehouse Station, NJ, USA; and Puregon, N.V. Organon, Oss, Netherlands) obtained from Chinese hamster ovary cells is used in humans, whereas follitropin and lutropin obtained from porcine pituitary extracts are used in animals (Table 3). Moreover, non-pituitary gonadotropins are commonly used in animals; they occur in products containing gonadotropin from a pregnant mare (eCG) or chorionic gonadotropin from pregnant women's urine (hCG) ( Table 3). The above shows that easily available gonadotropins from natural sources are used in animals.

Monoclonal Antibodies
The literature contains information on attempts to use monoclonal antibodies in animal therapy. Antibodies neutralizing beta-hemolysin produced by Staphylococcus aureus were used in cattle therapy [76]. Antibodies reducing the virulence of the avian flu virus (H5N1) were used in poultry treatment [77]. Neutralizing monoclonal antibody against flu virus A was used in pig treatment [78]. Antibodies against fibronectin A binding protein and against the clumping factor A were used in cattle treatment [79], which prevented the development of mastitis in cattle. Antibodies against infectious bursal disease virus (IBDV) were used in poultry treatment [80]. However, despite data suggesting the potential for therapeutic use of monoclonal antibodies in the treatment of selected animal diseases, there is still no market for commercially available products, with work on application of this group of drugs currently underway. Two monoclonal antibodies inhibiting the nerve growth factor (NGF) are undergoing clinical trials. These include ranevetmab (NV-01) for dogs and frunevetmab (NV-02) for cats, which are intended for the treatment of chronic pain and degenerative joint disease in cats and dogs [81].

Conclusions
In conclusion, the development of biotechnological methods provides new therapeutic opportunities; however, the number of drugs used in animal treatment introduced on the market is much smaller compared to drugs for human use. However, data suggest that the market will develop steadily and that new drugs are likely to be introduced in the near future for the treatment of skin diseases, inflammation of the muscular and articular system, cancers and immune diseases. The introduction of recombinant medicinal products, in particular, opens new therapeutic possibilities, which could not be possible with traditional allopathic therapy. This concerns, e.g., viral diseases such as canine parvovirus or feline FeLV and FIV, for which the recombinant interferon omega is the only drug registered in Europe. However, the application of biologic therapies is significantly limited by their high cost in the case of recombinant drugs. Moreover, further studies are necessary concerning the safety of these drugs. Moreover, further studies are necessary concerning the safety of these drugs. This is particularly important in the case of recombinant proteins, which may be characterized by high immunogenicity and could lead to undesirable results, e.g., anaphylactic shock.  Project financially co-supported by Minister of Science and Higher Education in the range of the program entitled "Regional lnitiative of Excellence" for the years 2019-2022, Project No. 010/RID/2018/19, amount of funding PLN 12,000,000.

Conflicts of Interest:
The authors declare no conflict of interest.