Microbial Testing And Its Impact On Non-Human Primates in Preclinical DMPK Studies

Preclinical Drug Metabolism and Pharmacokinetics (DMPK) studies are of paramount importance in the drug development process. These studies comprehensively characterize the Absorption, Distribution, Metabolism, and Excretion (ADME) processes of drugs within a biological system. Furthermore, they aid researchers in identifying potential drug-drug interactions and optimizing drug dosage and administration routes, significantly enhancing the success rates of subsequent clinical trials. DMPK studies also provide crucial data for evaluating potential drug toxicity and long-term safety, laying a robust foundation for clinical trials. Non-Human Primates (NHPs), due to their high degree of genetic, physiological, and pathological similarity to humans, play an irreplaceable role in DMPK research. This article aims to introduce NHP microbial testing programs and their impact on DMPK experimental outcomes, and to elaborate on the stringent measures Prisys Biotech takes in NHP quality standard control and quarantine procedures.

Mycobacterium tuberculosis (TB) is one of the most significant zoonotic pathogens in non-human primates, primarily transmitted via the respiratory and digestive tracts, exhibiting high infectivity. TB is mainly spread through droplets and aerosols produced when infected animals cough or sneeze. Furthermore, the urine, feces, and other secretions of infected animals may also carry the pathogen, contaminating the environment, feed, or water sources, leading to disease transmission. Monkeys infected with TB typically exhibit symptoms such as decreased appetite, weight loss, progressive emaciation, rapid or labored breathing, lethargy, and ruffled fur, potentially leading to death due to organ failure. Radiographic examinations often reveal signs of bronchopneumonia.

• Acute Cases: Disease progresses rapidly, sometimes with no obvious clinical signs before death.
• Chronic Cases: Hepatomegaly and splenomegaly may occur in the late stages.
• Progressive Cases: Local lymph node enlargement, and even ulceration of superficial lymph nodes.

Pathological examination often reveals disseminated millet-like nodules (0.5-3 mm in diameter) within the lung lobes, with characteristic caseous necrosis upon sectioning. Due to the similarity in symptoms between TB infection in monkeys and humans, TB control is crucial in NHP management. Strict quarantine measures, regular screening, and isolation of infected animals are key strategies to prevent TB transmission.

Cercopithecine Herpesvirus 1 (CHV-1), also known as B virus, naturally infects macaques, which serve as its natural reservoir. Macaques infected with B virus are typically asymptomatic carriers, although a minority may develop oral or genital ulcers or vesicles. B virus is primarily present in macaque saliva, blood, and other bodily fluids. NHPs infected with B virus may exhibit subtle changes in physiological status, including fluctuations in immune responses, minor weight variations, and slightly reduced appetite. Studies indicate that crab-eating macaques seropositive for B virus antibodies exhibit abnormalities in multiple biochemical parameters. For instance, levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and total bilirubin (TBIL) are higher in seropositive monkeys compared to normal monkeys, with TBIL showing statistically significant differences (P<0.05). Conversely, white blood cell count (WBC), hemoglobin (HGB), and monocyte count (MONO) levels are lower in seropositive monkeys, with MONO showing statistically significant differences (P<0.05). These subclinical physiological abnormalities may further influence drug ADME processes, potentially confounding the accuracy of DMPK study results. Human infection with B virus may initially present with flu-like symptoms, including fever, headache, and muscle pain. If left untreated, the virus can lead to severe neurological complications and even death. Although human cases of B virus infection are relatively rare, given the potential for serious clinical consequences and high mortality rates, continuous monitoring and preventive measures are crucial.

Salmonella spp. and Shigella spp. are common enteric pathogens, primarily transmitted via the fecal-oral route, and are zoonotic. Infections with these two bacteria can negatively impact the health of laboratory animals and the reliability of experimental results. NHPs infected with Salmonella spp. or Shigella spp. may exhibit symptoms such as diarrhea, mucoid bloody stools, abdominal pain, elevated body temperature, and loss of appetite. Shigella spp. infections are more prevalent in NHPs. Industry data indicate that the annual detection rate of Shigella spp. can be as high as 6-16%. Animals may carry these pathogens asymptomatically, but disease may be triggered under stressful conditions such as transportation, quarantine, and breeding seasons. NHPs infected with Salmonella spp. or Shigella spp. can typically be effectively controlled with antibiotic treatment for 5-7 days, with post-treatment re-testing usually yielding negative results. However, preventive measures remain crucial, including regular screening and monitoring, improving hygiene in animal housing environments, minimizing stress factors, and strictly implementing quarantine and inspection procedures. Both Salmonella spp. and Shigella spp. can be transmitted to humans through direct contact or contaminated environments, causing similar gastrointestinal symptoms. Therefore, strict biosafety measures must be implemented in NHP management to protect the health of laboratory personnel.

In addition to the aforementioned zoonotic pathogens that must be excluded, three retroviruses warrant special attention: Simian Immunodeficiency Virus (SIV), Simian Retrovirus (SRV), and Simian T-lymphotropic Virus (STLV). Although these viruses are not required to be excluded in SPF grade NHPs according to national standards, they can propagate within monkey colonies and establish persistent infections, potentially leading to abnormal clinical symptoms, altered physiological parameters, and biochemical responses in monkeys, thus potentially interfering with DMPK study outcomes.

Simian Immunodeficiency Viruses (SIV) belong to the Lentivirus genus and are closely related to Human Immunodeficiency Virus (HIV). Their natural hosts are primarily African monkeys and apes, where infections are often subclinical. However, when transmitted to Asian primates, SIV can induce immunodeficiency diseases resembling AIDS. Monkeys infected with SIV may exhibit a range of clinical signs, including chronic diarrhea, weight loss, and opportunistic infections. Furthermore, SIV infection can lead to impaired immune system function, thereby affecting drug metabolism and efficacy, potentially confounding the interpretation of DMPK study results. Studies suggest that SIV infection can cause abnormalities in hematological and serum biochemical parameters, which may introduce confounding variables into pharmacokinetic experiments.

Simian Retrovirus (SRV), also known as Simian Type D Retrovirus, belongs to the Betaretrovirus genus and primarily infects Asian macaques (such as rhesus macaques and cynomolgus macaques). SRV exists in multiple serotypes, with varying infection rates across different macaque populations. It can spread within monkey colonies through horizontal transmission (e.g., saliva contact, contaminated equipment contact) and vertical transmission (transplacental transmission). SRV infection may suppress the function of T lymphocytes and B lymphocytes. Histopathological studies indicate that SRV infection may induce ectopic lymphoid hyperplasia in the kidneys or bone marrow. Additionally, SRV infection can lead to hematological abnormalities, such as anemia and granulocytopenia. Literature reports indicate that between 1996 and 2002, at least 8 studies using rhesus or cynomolgus macaques were affected by SRV infection, with NHPs in 5 of these studies exhibiting anemia (hematocrit <25%). These abnormalities may cast doubt on the validity of experimental data and even necessitate the repetition of parts of studies, increasing research costs and timelines.

Simian T-lymphotropic Virus (STLV) belongs to the Deltaretrovirus genus and is related to Human T-cell Lymphotropic Virus (HTLV). Natural hosts of STLV include various African and Asian monkeys and apes, and it is endemic in non-human primate populations. STLV is a highly cell-associated virus with tropism for CD4+ and CD8+ lymphocytes. Transmission routes include breeding, lactation, or bites. Most infections are typically asymptomatic, but in some cases, STLV can induce T-cell lymphoma or lymphoproliferative diseases and may possess immunostimulatory and potentially immunosuppressive effects, as well as alter cytokine expression. Compared to uninfected NHPs, STLV-infected NHPs exhibit significantly elevated levels of IFN-γ and IL-2. STLV infection can lead to complex changes in the immune system, increasing susceptibility to other pathogens and potentially interfering with the results of pharmacokinetic experiments.

NHPs used at Prisys Biotech, in addition to meeting the national SPF grade NHP standards, are further rigorously required to test negative for Simian Immunodeficiency Virus (SIV), Simian Retrovirus (SRV), and Simian T-lymphotropic Virus (STLV). This higher standard of NHPs is commonly referred to in the industry as "five-negative monkeys," meaning negative for five pathogens: Mycobacterium tuberculosis (TB), Cercopithecine Herpesvirus 1 (BV), SIV, SRV, and STLV.

To ensure NHP quality, Prisys Biotech implements stringent audits and supervision of NHP sources and conducts comprehensive quarantine work. Upon arrival at our facilities, NHPs undergo thorough microbial testing, complete blood counts, and serum biochemical analyses during the quarantine period to ensure their health status fully meets experimental requirements. NHPs in DMPK studies typically require long-term housing; therefore, we implement the following multiple measures to safeguard the health of our monkey colonies:

• Regular Comprehensive Health Checks: Regular comprehensive health checks are performed on the colony to continuously monitor their health status.
• Hematological Screening Before IND Projects: Prior to the initiation of Investigational New Drug (IND) projects, comprehensive hematological examinations are conducted to further screen for high-quality NHPs.
• Pre-Project Physical Condition Checks: Before the initiation of all DMPK projects, meticulous physical condition checks are performed on NHPs to re-confirm their health status.
• Veterinary Assessment and Treatment for Abnormal Animals: For animals exhibiting abnormal health conditions, experienced veterinary teams provide professional assessment and timely treatment.
• Adequate Drug Washout Period Post-Project: After project completion, NHPs are provided with a sufficient drug washout period to ensure animals recover to a healthy state before being used in subsequent pharmacokinetic (PK) experiments.

Furthermore, to enhance animal welfare and minimize NHP stress responses, Prisys Biotech actively employs positive reinforcement training methods to acclimate NHPs. Restraint and blood collection are among the most common procedures in DMPK experiments, and NHPs, being inherently sensitive, are prone to significant stress responses during these procedures. Studies have shown that training can significantly reduce stress levels in monkeys. We have comprehensively implemented training methods for monkey restraint and blood collection, effectively reducing stress during experiments and simultaneously lowering the potential risk of injury to both personnel and animals during experimental procedures.

Conclusion

Preclinical DMPK studies play a pivotal role in innovative drug development. NHPs, as a critical animal model, have their health and quality directly impacting the reliability and reproducibility of research results. Prisys Biotech consistently upholds high-quality industry standards for NHPs. The NHPs used in our DMPK studies are all "five-negative monkeys," providing a solid guarantee for the reliability of experimental results. Moreover, through our unique restraint and blood collection training methods, we can effectively reduce stress responses in NHPs, ensuring the accuracy and reproducibility of experimental outcomes, thereby better facilitating innovative drug research and accelerating the drug development and market launch process.