ABSTRACT
Background
Considering unmet clinical needs in the dementia therapeutics world, PCMCV002 was developed as a cheap and easily accessible adjunct therapy option. It is a combination of amino acids, vitamins, and essential nutrients formulated in a best-fit complex medicinal food supplement as an adjunct therapy to the already established dementia therapies.
Aim
The aim of this study is to evaluate the sub-acute preclinical toxicity profile of PCMCV002 in Wistar rats.
Materials/Method
PMCV002, ketamine, Photoelectric colorimeter model AE 11D (Erma Inc. Japan), Hematology analyzer (Sysmex USA), hematocrit, centrifuge, spectrophotometer, weighing balance, cotton wool, scissors, animal cages, plastic containers, plain and heparinized plastic bottles. The study involved hematology, hepatorenal and electrolytes changes and histological evaluation 28 days postdosing daily with PCMCV002 using OECD test Guideline 407 (2008).
Conclusions
The findings following a 28-day administration of PMCV002 revealed that it is relatively nontoxic in rats, as no obvious harmful effects were noted on hematological and biochemical indices. The histo-architecture of the brain, heart, liver, and kidneys following a 28-day administration of PCMCV002 to the test rats did not show any significant pathologic changes. All these indicate a quite safe medicinal product, coupled with maximum tolerable dose as high as 5000 mg/kg.
KEYWORDS: Preclinical, toxicology, adjunct, dementia, food supplement, medicinal product
1. Introduction
Millions of people globally have dementia. It is a progressive neurodegenerative disorder characterized by significant brain loss due to the accumulation of amyloid plaques and neurofibrillary tangles in the human brain. As of 2015, an estimated 46 million individuals worldwide were believed to suffer from dementia; this estimate is projected to increase to 131.5 million by 2050 [1].
The treatment options available at present include cholinesterase inhibitors, anti-glutamates, antidepressants, monoclonal antibody therapies, and antipsychotics as disease-modifying agents to delay disease progression and manage dementia’s dire symptoms [2]. In terms of specifics, there are only six approved and five established treatments for cognitive symptoms of dementia in the UK. Three of them are anticholinesterases [3] – Donepezil (Aricept), Galantamine (Razadyne), and Rivastigmine (Exelon), which is the go-to drug when dementia patients can’t tolerate it orally, as it is administered as a transdermal patch. These 3 drugs belong to the class of drugs known as anticholinergics, and they exert their therapeutic effect by enhancing cholinergic function. This is accomplished by increasing the concentration of acetylcholine at the synapse through reversible inhibition of their hydrolysis by cholinesterase [4]. These drugs are generally well tolerated but can cause side effects such as nausea, vomiting, loss of appetite, and increased frequency of bowel movements [5].
A fourth drug, Memantine (Namenda), is a common dementia drug used in the UK; it modulates glutamate activity by low to moderate affinity uncompetitive (open-channel) NMDA receptor antagonism, preferentially binding to the NMDA receptor-operated cation channels [6]. This can cause side effects, including headache, constipation, confusion, and dizziness [7]. Memantine and Donepezil are available in a combined formulation branded Nazerac as a fifth established drug for dementia. Relatively new to the scene of dementia therapeutics is Aducanumab [8]. It has since been withdrawn from clinical use for lack of efficacy. Like Adulizimab, two other similar agents had completed Phase 3 clinical trials on demented patients as anti-amyloid beta (Aβ) monoclonal antibodies (mAb). These are Biogen/Eisai drug lecanemab (Leqembi) and the Lilly drug, donanemab. Donanemab was fast-tracked by the United States Food and Drug Administration (FDA), and lecanemab later received full approval for the treatment of AD [9], but they have both failed to provide meaningful benefits to demented patients and improvements in cognitive symptoms [9,10]. Thus, there is a clear need for new, accessible adjunct treatments for dementia patients. PCMCV002 therefore holds such potential as a cost-effective adjunct treatment for cognitive symptoms of dementia from the preclinical efficacy cognitive impairment rats modeled studies done so far, using an alternative mechanism of action. This would be well described in part 2 of the 3-phase study to be published as a follow-up to this article. When PCMCV002 is successfully trialed, as “best-fit combination of essential amino acids, vitamins and nutrients, it would serve as an easily accessible food supplement and adjunct therapy to dementia care.”
Previously, seven amino acids in a granular powder were studied to see if the combination can delay the onset of dementia [11]. Protein malnutrition is well documented as a potential risk factor for senile dementia development in the elderly age group, though the precise link between protein/amino acid lacks and cognitive impairment is yet to be fully understood. The trial revealed that the 7-amino acid combination therapy showed significantly improved cognitive function (Trail Making Test B), social interaction, and psychological health scores after ingestion compared to the control [11].
The result of LP7 study acted as a platform for us to establish our hypothesis to develop PCMCV002 as combination therapy comprising L-Citrulline as an amino acid with a guanidino group appended to a standard amino acid framework for its role as a powerhouse reservoir of nitric oxide in addition to another vital amino acid, glycine. Though more studies would be required to prove this, there is a strong possibility that adequate nitric oxide levels in the brain help with the preservation of learning and memory, with the overall benefit hypothesized to be a reduction in the number of beta-amyloid plaques and tau phosphorylation in the hippocampus, as suggested by previous studies which showed that nitric oxide levels decline evident in Alzheimer’s disease patients [12].
Glycine was chosen as a key component of PCMCV002 to combat protein malnutrition, which underlies senile dementia and reduces muscle loss due to its ability to aid the production of creatine and collagen in humans. Glycine is also known to boost short-chain fatty acids (SCFAs) production, which is beneficial for improving cardiometabolic functions and gut microbiome to improve cognition invariably. Like L-Citruline, it also possesses the ability to protect brain cells from oxidative stress damage from free radicals by producing glutathione and improve the ability of the body to utilize nitric oxide [13,14].
In addition to glycine, PCMCV202 was fortified with folic acid. A study in 2002 published in Journal of Neurosciences showed mice with diet fortified with folic acid had a higher number of viable counted neurons in their hippocampus compared to those fed with folic acid deficient diet [15]. Vitamin E was also added to PCMCV002 formulation as a historic Alzheimer’s Disease Cooperative study published in the New England Journal of Medicine in 1997 revealed that moderate to severe cognitively impaired patients who were dosed with 2000 IU daily of Vitamin E (Alpha tocopherol) or selegiline or both had slower progression of disease in terms of losing the ability to independently dress, bathe, and use the toilet themselves compared to the group who did not take Vitamin E daily [16,17]. Green Coffee Extract, for its caffeine content and the latter’s beneficial role in reducing cognitive decline, was also included in the PCMCV002 formulation. Caffeine, due to its high fiber content, can stimulate gastric hydrochloric acid production to promote increased bowel motions to combat chronic constipation, which is a key feature seen in demented patients. The combination of increased bowel motion, change in the gut microbiome to affect the Gut-Brain axis to improve cognition, and ability to provide essential nitric oxide to reduce oxidative damage and mop up free radicals are the key mechanisms of action PCMCV002 expected to act on to exert its cognition improvement action. All these were put into consideration in formulating PCMCV002 as a combination effervescent tablet and oral thin film strip formulations containing L-Citrulline, glycine, Vitamin E, Folic acid, Green Coffee Extract, and standard industry excipients. L-Citrulline is the key active agent in the food supplement PCMCV002, it is a neutral amino acid and a major precursor of L-arginine in the nitric oxide (NO) cycle. It has been previously studied and documented to cross the blood-brain barrier (BBB) using the conditionally immortalized rat brain capillary endothelial cell line (TR-BBB cells), as an in vitro model of the BBB [18].
2. Materials and methods
PMCV002, ketamine, Photo electric colorimeter model AE 11D (Erma Inc. Japan), Hematology analyzer (Sysmex USA), hematocrit, centrifuge, spectrophotometer, weighing balance, cotton wool, scissors, animal cages, plastic containers, plain and heparinized plastic bottles.
2.1. Experimental animals
Male and female Wistar rats weighing 100–150 g were obtained from the experimental Animal House Facility, Faculty of Pharmaceutical Sciences, Ahmadu Bello University Zaria. The rats were maintained in a well-ventilated room and given access to water and standard feed ad libitum. The study was carried out as per the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. Healthy animals and non- pregnant females were included. Animals of weights less than 100 g were excluded.
2.2. Acute toxicity study
The median lethal dose (LD50) of PMCV002 was established in accordance with the Organization for Economic Co-operation and Development (OECD, 425) Guideline [19] using five (5) Wistar rats which were fasted overnight before dosing with PMCV002 at the dose of 5000 mg/kg orally. One (1) rat was administered the test agent and monitored for the first 24 hours and then for 14 days for signs of toxicity and death. The remaining four (4) rats were also administered the same dose and observed for 2 weeks for toxic effects and mortality.
2.3. Sub-acute toxicity study
A sub-acute toxicity study was conducted using the OECD test Guideline 407 [20]. Thirty- two (32) rats were randomly distributed into four (4) groups (three treatment groups and one control group) of eight (8) animals each (4 males and 4 females) in different cages.
The rats in the control group were given distilled water (1 mL/kg), and those in the treatment groups were administered graded doses (1000, 500, and 250 mg/kg) of the PMCV002 for four (4) weeks using oral gavage. The rats and toxic effects. Their feed intake, water intake and body weights were checked weekly throughout the study period. On day twenty-nine (29), the animals were euthanized using ketamine (90 mg/kg) to anaesthetize them [21]. Blood samples were collected from jugular veins into heparinized and plain bottles. The livers, kidneys, stomachs, lungs, brains, and hearts were collected into sample containers. The organs were weighed (dual organs were weighed together). The relative organ-body weight ratio was subsequently evaluated.
2.4. Evaluation of hematological parameters
The blood samples collected into ethylene diamine tetra-acetic acid containing sample bottles were used to analyze packed cell volume (PCV), red blood cells (RBC), white blood cells (WBC), platelets (PLT), lymphocytes (LYMP), and granulocytes (GRAN) using an auto-hematology analyzer (Sysmex USA) [22].
2.5. Evaluation of biochemical parameters and electrolytes
The blood samples collected into plain bottles were centrifuged for 10 minutes at 3500 rpm. The serums derived following centrifugation were used for the assessment of biochemical indices which include alkaline phosphatase (ALP) [23], aspartate amino transferase (AST) and alanine amino transferase (ALT) [24], total bilirubin [25], albumin and total protein [26], creatinine, and urea [27]. Electrolytes such as chloride, bicarbonate, potassium, and sodium were also analyzed using established protocols.
2.6. Histological evaluation
The livers, kidneys, stomachs, lungs, brains, and hearts were fixed in 10% formaldehyde for ten (10) days. They were further processed to get 5 μm sections of the various tissues before staining with hematoxylin and eosin. The stained sections were viewed under microscope for histo-morphological alterations [28,29].
2.7. Data analysis
Data obtained were presented as mean ± standard error of the mean (S.E.M.). Comparisons between means of the treatment groups were carried out using either One Way or Repeated Measures Analysis of variance (ANOVA). The results were presented as tables.
3. Results
3.1. Acute toxicity profile of PMCV002 in rats
The administration of PMCV002 at 5000 mg/kg did not produce any obvious sign of toxicity and death throughout the observation period of fourteen (14) days. Hence, the median lethal dose was estimated to be above 5000 mg/kg in rats.
3.2. Effect of PMCV002 on feed intake of rats following 28-day oral administration
The administration of PMCV in both male and female rats did not produce significant (p > 0.05) changes in feed intake compared to control and over time (Supplementary Table S1).
3.3. Effect of PMCV002 on water intake of rats following 28-day oral administration
The administration of PMCV002 in both male and female rats did not produce significant (p > 0.05) changes in water consumption compared to control and over time (Supplementary Table S2).
3.4. Effect of PMCV002 on body weight of rats following 28-day oral administration
The administration of PMCV002 for four (4) weeks did not produce any significant effect in the body weights of rats compared to the control (p > 0.05), but the PMCV002 at the doses of 250 and 500 mg/kg produced a significant increase (p < 0.05) in body weight of male rats in weeks 3 and 4 compared to week 1. In the female rats, PMCV002 at 250 mg/kg showed a significant increase (p < 0.05) in week 4 compared to week 1 (Table 1).
Table 1.
Effect of PMCV002 on body weight of rats following 28-day oral administration.
| Mean body weight (g) |
||||
|---|---|---|---|---|
| Treatment (mg/kg) | Week 1 | Week 2 | Week 3 | Week 4 |
| Male | ||||
| D/W 1 mL/kg | 112.25 ± 7.69 | 115.25 ± 9.28 | 119.40 ± 8.10 | 129.40 ± 8.81 |
| PMCV (1000) | 127.40 ± 9.63 | 120.80 ± 8.18 | 120.80 ± 6.98 | 140.60 ± 9.38 |
| PMCV (500) | 125.20 ± 8.74 | 136.60 ± 9.29 | 147.60 ± 8.43a | 146.00 ± 6.73a |
| PMCV (250) | 110.40 ± 9.41 | 117.00 ± 6.80 | 132.80 ± 6.55a | 145.25 ± 8.70a |
| Female | ||||
| D/W 1 mL/kg | 109.40 ± 9.75 | 114.80 ± 7.55 | 112.00 ± 9.00 | 119.40 ± 7.18 |
| PMCV (1000) | 110.25 ± 9.04 | 110.75 ± 5.37 | 112.75 ± 5.56 | 114.50 ± 5.58 |
| PMCV (500) | 127.00 ± 7.66 | 137.20 ± 9.31 | 132.75 ± 7.57 | 139.50 ± 5.60 |
| PMCV (250) | 124.50 ± 7.71 | 125.80 ± 8.85 | 136.60 ± 7.78 | 144.40 ± 7.89a |
Values are Mean ± S.E.M. a=p < 0.05, compared to Week 1 – Repeated Measures ANOVA followed by Bonferroni post hoc test, n = 4 (Male or female), D/W = Distilled water.
3.5. Effect of PMCV002 on relative organ-weight of rats following 28-day oral administration
The administration of PMCV002 did not produce any significant (p > 0.05) increase or decrease in the relative organ/body weights of the male and female rats in comparison with the control (Table 2).
Table 2.
Effect of PMCV002 on relative organ-weight of rats following 28-day oral administration.
| Treatment (mg/kg) |
||||
|---|---|---|---|---|
| Parameters | D/W 1 mL/kg | PMCV 1000 | PMCV 500 | PMCV 250 |
| Male | ||||
| Liver | 3.45 ± 0.05 | 3.15 ± 0.05 | 2.45 ± 0.25 | 3.15 ± 0.15 |
| Kidney | 0.70 ± 0.00 | 0.70 ± 0.10 | 0.50 ± 0.00 | 0.60 ± 0.00 |
| Stomach | 0.95 ± 0.05 | 1.20 ± 0.30 | 1.00 ± 0.10 | 0.70 ± 0.10 |
| Brain | 0.75 ± 0.05 | 1.00 ± 0.00 | 0.90 ± 0.10 | 0.90 ± 0.10 |
| Heart | 0.15 ± 0.00 | 0.45 ± 0.50 | 0.30 ± 0.00 | 0.40 ± 0.00 |
| Lungs | 0.87 ± 0.20 | 0.60 ± 0.05 | 0.780 ± 0.00 | 0.55 ± 0.05 |
| Female | ||||
| Liver | 2.89 ± 0.15 | 2.98 ± 0.05 | 3.50 ± 0.10 | 3.86 ± 0.15 |
| Kidney | 0.35 ± 0.00 | 0.54 ± 0.00 | 0.79 ± 0.00 | 0.71 ± 0.05 |
| Stomach | 0.65 ± 0.00 | 1.14 ± 0.20 | 0.77 ± 0.05 | 1.15 ± 0.05 |
| Brain | 0.84 ± 0.05 | 1.20 ± 0.00 | 0.80 ± 0.05 | 1.10 ± 0.05 |
| Heart | 0.36 ± 0.05 | 0.39 ± 0.05 | 0.46 ± 0.05 | 0.43 ± 0.00 |
| Lungs | 1.39 ± 0.10 | 1.85 ± 0.05 | 1.70 ± 0.05 | 1.36 ± 0.05 |
Values are Mean ± S.E.M; No significant differences between groups – One way ANOVA, n = 2 (Male or Female), D/W = Distilled water.
3.6. Effect of PMCV002 on hematological parameters of rats following 28-day oral administration
The administration of PMCV002 in both male and female rats for 28 days did not produce any significant (p > 0.05) changes in the hematological indices when compared to the control (Table 3).
Table 3.
Effect of PMCV002 on hematological parameters of rats following 28-day oral administration.
| Treatment (mg/kg) | WBC (×103/µL) |
RBC (×106/µL) |
PCV (%) |
Platelets (×103/µL) |
Granulocytes (%) |
Lymphocytes (%) |
|---|---|---|---|---|---|---|
| Male | ||||||
| DW (1 mL/kg) | 4.37 ± 1.11 | 5.93 ± 0.03 | 36.00 ± 2.00 | 221.67 ± 23.67 | 2.93 ± 0.59 | 6.90 ± 0.35 |
| PMCV (1000) | 4.93 ± 0.88 | 6.00 ± 0.00 | 36.33 ± 0.67 | 182.00 ± 7.94 | 2.80 ± 0.58 | 6.20 ± 0.40 |
| PMCV(500) | 4.17 ± 0.32 | 5.60 ± 0.30 | 34.00 ± 0.00 | 187.00 ± 1.00 | 2.47 ± 0.67 | 6.57 ± 0.64 |
| PMCV(250) | 3.87 ± 0.30 | 6.10 ± 0.10 | 35.33 ± 0.88 | 184.33 ± 2.91 | 2.53 ± 0.27 | 5.40 ± 0.76 |
| Female | ||||||
| DW (1 mL/kg) | 4.33 ± 0.78 | 5.70 ± 0.31 | 36.67 ± 1.45 | 190.37 ± 11.99 | 2.17 ± 0.32 | 6.17 ± 0.55 |
| PMCV (1000) | 5.63 ± 0.82 | 6.10 ± 0.12 | 37.67 ± 1.86 | 231.67 ± 33.17 | 2.37 ± 0.43 | 6.23 ± 0.17 |
| PMCV(500) | 4.33 ± 0.43 | 5.87 ± 0.03 | 39.00 ± 3.06 | 179.00 ± 12.49 | 2.60 ± 0.25 | 5.83 ± 0.35 |
| PMCV(250) | 5.37 ± 0.47 | 5.70 ± 0.30 | 37.33 ± 0.88 | 182.03 ± 13.51 | 2.47 ± 0.28 | 5.10 ± 0.52 |
Values are Mean ± S.E.M; No significant differences as compared to DW group – One-Way ANOVA followed by Bonferroni’s post hoc test, n = 4, DW = Distilled water, WBC = White blood cells, RBC = Red blood cells, PCV = Packed cell volume.
3.7. Effect of PMCV002 on liver parameters of rats following 28-day oral administration
The administration of PMCV002 in both male and female rats for 28 days did not produce any significant (p > 0.05) changes in the hepatic indices when compared to the control (Table 4).
Table 4.
Effect of PMCV002 on liver parameters of rats following 28-day oral administration.
| Treatment (mg/kg) | ALT (×103/µL) |
AST (×106/µL) |
ALP (%) |
Total protein (×103/µL) |
Albumin (%) |
|---|---|---|---|---|---|
| Male | |||||
| DW (1 mL/kg) | 11.00 ± 2.08 | 44.67 ± 9.78 | 28.90 ± 5.82 | 9.20 ± 2.13 | 1.77 ± 0.38 |
| PMCV (1000) | 15.67 ± 1.76 | 48.00 ± 4.73 | 26.43 ± 4.76 | 10.53 ± 1.86 | 1.70 ± 0.10 |
| PMCV(500) | 18.67 ± 5.93 | 43.00 ± 9.07 | 28.43 ± 6.07 | 11.03 ± 2.60 | 2.23 ± 0.28 |
| PMCV(250) | 12.33 ± 2.91 | 33.00 ± 7.02 | 26.17 ± 7.25 | 10.73 ± 4.29 | 2.07 ± 0.48 |
| Female | |||||
| DW (1 mL/kg) | 10.67 ± 2.33 | 54.33 ± 7.51 | 19.13 ± 1.02 | 6.50 ± 0.64 | 1.90 ± 0.29 |
| PMCV (1000) | 9.00 ± 1.53 | 36.67 ± 0.33 | 19.67 ± 0.90 | 6.37 ± 0.37 | 1.27 ± 0.18 |
| PMCV(500) | 12.00 ± 1.53 | 47.00 ± 1.73 | 24.37 ± 5.5 | 6.40 ± 0.60 | 2.33 ± 0.34 |
| PMCV(250) | 16.00 ± 2.08 | 57.67 ± 5.81 | 25.23 ± 5.32 | 6.90 ± 0.67 | 1.70 ± 0.12 |
Values are Mean ± S.E.M; No significant differences as compared to DW group – One-Way ANOVA n = 4, DW = Distilled water, ALT = Alanine amino transferase, AST = Aspartate amino transferase, ALP = Alkaline Phosphatase.
3.8. Effect of PMCV002 on kidney parameters of rats following 28-day oral administration
The administration of PMCV002 in both male and female rats for 28 days did not produce any significant (p > 0.05) changes in the renal indices and electrolytes when compared to the control (Table 5).
Table 5.
Effect of PMCV002 on kidney function parameters of rats following 28-day oral administration.
| Treatment (mg/kg) | Urea (mg/dL) |
Creatinine (mEq/L) |
Sodium (mmol/L) |
Potassium (mmol/L) |
Chloride (mg/dL) |
Bicarbonate (mg/dL) |
|---|---|---|---|---|---|---|
| Male | ||||||
| DW (1 mL/kg) | 81.67 ± 6.67 | 1.17 ± 0.17 | 189.03 ± 14.86 | 13.53 ± 4.23 | 38.33 ± 3.84 | 101.33 ± 4.33 |
| PMCV (1000) | 136.67 ± 10.93 | 0.77 ± 0.17 | 231.23 ± 6.23 | 16.90 ± 4.28 | 32.33 ± 4.37 | 81.67 ± 8.82 |
| PMCV(500) | 97.00 ± 7.00 | 0.87 ± 0.09 | 209.10 ± 19.40 | 14.17 ± 4.19 | 28.00 ± 1.00 | 94.33 ± 7.06 |
| PMCV(250) | 111.67 ± 23.33 | 0.83 ± 0.09 | 229.13 ± 19.97 | 13.57 ± 3.91 | 32.67 ± 2.33 | 117.33 ± 14.33 |
| Female | ||||||
| DW (1 mL/kg) | 92.00 ± 6.61 | 1.00 ± 0.15 | 123.50 ± 46.07 | 11.23 ± 1.65 | 44.67 ± 7.86 | 101.00 ± 2.00 |
| PMCV (1000) | 100.00 ± 5.77 | 1.03 ± 0.07 | 196.83 ± 51.64 | 13.10 ± 1.08 | 49.00 ± 8.72 | 108.67 ± 3.67 |
| PMCV(500) | 82.33 ± 3.93 | 0.80 ± 0.00 | 116.27 ± 28.08 | 11.67 ± 1.92 | 39.00 ± 3.61 | 109.33 ± 4.93 |
| PMCV(250) | 101.67 ± 6.01 | 1.10 ± 0.05 | 103.13 ± 35.50 | 15.40 ± 3.89 | 37.00 ± 1.15 | 95.00 ± 4.16 |
Values are Mean ± S.E.M; No significant differences as compared to DW group – One-Way ANOVA, n = 4, DW = Distilled water.
3.9. Effect of PMCV002 on the histology of the liver, kidney, heart, and brain of rats following 28-day oral administration
Histological examination of the liver sections showed morphological changes in PMCV treated groups of both the male and female rats manifested as vacuolation and hepatocellular necrosis (Figure 1). Histological investigation of the kidney sections revealed moderate tubular necrosis, tubular adhesion, and lymphocyte hyperplasia (Figure 2). However, there were no significant structural changes in the histo-architecture of the heart and brain (Figures 3 and 4) respectively.
Figure 1.
Distilled water treated group showing normal hepatocytes; PMCV002 1000 mg/kg treated group showing slight hepatocellular necrosis (HN) and vacuolation (V), PMCV002 500 mg/kg treated group showing slight hepatocellular necrosis (HN) and vacuolation (V), PMCV002 250 mg/kg treated group showing normal hepatocytes in males and vacuolation (V) in females (H and E, magnification × 250).
Figure 2.
Distilled water treated group showing normal tubules and glomerulus; PMCV 1000 mg/kg treated group showing lymphocyte hyperplasia (LH); PMCV 500 mg/kg treated group showing tubular adhesion (TA) and PMCV 250 mg/kg treated group showing slight tubular necrosis (TN) (H and E, magnification × 250).
Figure 3.
All groups showing normal features (H and E, magnification × 250).
Figure 4.
All groups showing normal features (H and E, magnification × 250).
4. Discussion
PMCV002, as a medicinal food supplement product in development, is made of refined “best-fit” complex of vitamins and amino acids, which are proven safe [30]. Thus, PCMCV002 after 28 days of dosing in test Wistar rats was subjected to hematologic, hepatorenal, and histopathologic toxicity studies. Toxicological studies are essential in drug discovery and development because they bridge the gap between experimental findings and risk assessment and help in curtailing adverse drug reactions [31].
Five (5) Wistar rats were administered with PCM002 at 5000 mg/kg to establish preliminary toxicological profile and selection of doses for the 28-day studies [32,33]. The non-mortality in the rats implies the lethal dose of PCMCV002 is above 5000 mg/kg. This would be beneficial to demented patients, as an overdose, if it occurs accidentally, won’t put the users of PCMCV002 at grave risk.
Changes in feed and water intake in relation to body weight increase provide a reliable information on the overall health condition of experimental animals [34]. In this study, there were no obvious changes in feed and water intake relative to the control (Supplementary Tables S1 and S2). This implies that PMCV002 does not impair utilization of food by the experimental animals.
Drastic changes in body weights as well as internal organ weights indicate toxicity and are important parameters in assessing the safety of drugs and xenobiotics [35]. Weight loss or weight gain in toxicological studies can be seen as a sign of a toxic experimental agent. PMCV002 did not produce any worrisome effect on the body weights of rats compared to
the control (p > 0.05). However, PMCV002 at the doses of 250 and 500 mg/kg produced a significant increase (p < 0.05) in the body weight of male rats in weeks 3 and 4 compared to week 1. In the female rats, PMCV002 at 250 mg/kg showed a significant increase (p < 0.05) in week 4 compared to week 1 (Table 1). However, this increase in body weight is seen as a beneficial effect of the investigational therapeutic agent as it was not too extreme. The effect of PMCV002 on the relative organ-weight of Rats following 28-day oral administration (Table 2) wasn’t marked, signifying non-deleterious effects on the organs.
Investigation of the hematological parameters gives an idea of the extent of toxicity caused by drugs and xenobiotics in animals [36]. The test rats dosed with PCMCV002 were also evaluated for hematologic toxicity looking at anemia, neutropenia, lymphopenia, and thrombocytopenia following its 28-day course treatment. None of these were evident (Table 3).
Acute hepatorenal toxicity is one of the biggest reasons why drugs fail in human trials when developing new therapeutic agents. The liver plays a significant role in the metabolism nutrients and removal of toxins from the body [37] and a rise in the levels of serum transaminases implies hepatotoxicity [38]. In this study, we looked for drastic pathologic changes in the levels of ALP, AST, and ALT following the administration of PMCV002, and none was evident (Table 4). The serum urea and creatinine levels are usually elevated when the kidney tissues have been damaged and functions hindered [39]. In this study, PMCV002 did not significantly increase the urea and creatinine levels (Table 5). Non-elevation in the level of creatinine suggests that the agent does not affect renal function.
Histopathology is widely acknowledged as a critical part of the toxicologic and risk assessment, and it provides information on potential safety issues associated with new drugs and molecules under development [40,41]. Histological investigation of the liver, the major organ involved in the metabolism of drugs and xenobiotics, showed no severe structural distortions in its histo-morphology following a 28-day administration of PMCV002 (Figures 1–4) [42]. Similarly, the kidney, heart, and brain showered no histopathological features. The agent under investigation (PMCV002) crosses the blood-brain barrier and the next phase of its preclinical evaluation would be the efficacy study using dementia-disease model (scopolamine-induced amnesia) in rats and evaluating relevant memory loss biomarkers; Beta Amyloid 42, Phospho tau 181 and 205, serum homocysteine, brain-derived neurotrophic factor (BDNF), and nerve growth factor (NGF). Thereafter, we will proceed to human demented patients administered PCMCV002 in a controlled clinical trial setting for proper evaluation of its efficacy and toxicity profiles.
5. Conclusions
In conclusion, PCMCV002 was developed with vital therapeutic actions in mind following 28 days of daily dosing in memory-impaired model Wistar as a basis to postulate in demented humans it would combat protein malnutrition, which underlines senile dementia through the inclusion of key amino acids, change in gut microbial diversity of demented patients to beneficial commensal abundance to promote increased bowel motion, improve stool consistency, provide vital nitric oxide to combat oxidative stress, and mop up free radicals. We also had in mind to develop PCMCV002 as a medicinal product with a neurotrophic ability to preserve viable neurons in vital memory centers of the brain, as well as promote neurogenesis and exert a change in the gut microbiome to modify demented patients’ gut-brain axis to improve cognition.
At the end of the next phase of this study, which includes relevant dementia biomarkers and cognitive impairment behavioral tests, we would be able to determine if PCMCV002 can replicate the efficacy and nontoxic status seen in scopolamine-induced cognitive impairment in Wistar rats modeled in demented patients. We would also in the next studies use static blood-brain barrier (BBB) models in the form of monolayer BBB model made of endothelial cells grown in the Transwell to create a reproducible BBB model. This would be designed to mimic the blood side, whereas the well in which the insert fits would mimic the parenchymal side. It would have a microporous membrane support to allow the exchange of small molecules, but prevent the migration of cells between the two compartments to test the BBN crossing capability of PCMCV002.
The final study in the development of PCMCV002 as a potential adjunct dementia therapy would involve its test on human demented patients in the form of double-blinded randomized controlled clinical trial, and so potentially after human trial success, it can become an adjunct treatment tool to help with the huge void of unmet clinical needs of demented patients.
Supplementary Material
Acknowledgments
The authors wish to thank the Co-Director of Precisemed, Mr Daniel Onifade, for his contribution towards success of this study.
Funding Statement
This study was sponsored by Precisemed Limited UK.
Article highlights
PMCV002 does not have a deleterious effect on hematological and biochemical parameters of rats following 28-day administration.
PMCV002 does not have harmful effects on the brain, heart, liver, and kidneys of rats following 28-day administration.
Author contributions
AMA and AA contributed in study design, data acquisition, analysis, drafting and proofreading of the manuscript.
NAB and MAO participated in the experiment, data analysis and interpretation, drafting and proofreading of the manuscript.
AW, LAF and KW contributed to the experiment protocol, clinical implication of the study discussion and proofreading the manuscript.
Disclosure statement
AMA works for Precisemed Limited UK, the study sponsors who funded and provided PCMCV002 for the study. The authors have no other relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript apart from those disclosed.
No writing assistance was utilized in the production of this manuscript.
Reviewer disclosures
Peer reviewers on this manuscript have no relevant financial or other relationships to disclose.
Ethical disclosure
The studies were carried out with the approval of the Ahmadu Bello University Committee on Animal Use and Care (ABUCAUC) with the Ethical Approval Number ABUCAUC/2024/002.
Supplementary Information
Supplemental data for this article can be accessed online at https://doi.org/10.1080/20415990.2025.2545748
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