Acute Toxicity Evaluation of Proliferol: A Dose-Escalating,Placebo-Controlled Study in Rats

Abstract

Proliferol is an investigational new drug containing lidocaine hydrochloride 0.25%, dextrose 12.5%, glycerin 12.5%, and phe­nol 1.0% in aqueous solution. Despite extensive human experience with similar drugs administered by intraligamentous injection for chronic musculoskeletal disorders, little is known concerning preclinical toxicity. The purpose of this study was to assess the acute toxicity of intramuscular Proliferol in 96 (48 male, 48 female) Charles River strain rats, which were randomly assigned to low-(1×), medium- (5×), or high- (10×) dose Proliferol (derived from a human dose of 20 ml on a volume per bodyweight basis), or high-dose saline placebo. Observations included clinical observations, biochemistry, hematology, urinalysis, and full histopathology after 24 h or 14 days. There were no signs of ill health or reaction to treat­ment, and gait and body temperature were within normal limits. Biochemistry findings at 24 h included elevated aspartate aminotransferase, alanine aminotransferase, and haptoglobin; at 14 days all values were within normal ranges. Urinalysis findings at 24 h included increased urobilinogen and blood in all dose groups com­pared with placebo. Urine concentrations of phenol and lidocaine were greatest at 2 h and absent at 24 h. Histopathology findings included localized acute inflammatory soft tissue changes at the injection sites at 24 h and skeletal muscle regeneration at 14 days, which were consistent with the anticipated mechanism of action of Proliferol. There was no evidence of systemic toxicity from intra­muscular injection of Proliferol in rats at up to 10× the human dose.

Prolotherapy involves repeated intraligamentous injections of chemical irritant solutions intended to stimulate fibroblastic proliferation and connective tissue repair for chronic muscu­loskeletal and spinal pain due to suspected ligamentous injury (Banks 1991). A drug solution commonly used in prolotherapy contains lidocaine hydrochloride 0.25%, dextrose 12.5%, glycerin 12.5%, and phenol 1.0% in aqueous solution, and is supplied by compound pharmacies or prepared by physicians (Dagenais et al. 2006a). Although there is some evidence of clinical efficacy using this drug solution for spinal pain, methodological limitations preclude assigning the observed benefit to the drug injections and further clinical research is needed (Dagenais, Haldeman, and Wooley 2005). To support future clinical trials with this drug solution (recently named Proliferol), our group recently conducted a pilot preclinical acute toxicity study that attempted to mimic spinal intraligamentous drug administration currently performed in humans (Dagenais et al. 2006b). Methods from the pilot study were deemed fea­sible and a larger study was undertaken. The purpose of the present study was to assess the acute local and systemic toxicity of Proliferol in a dose-escalating, placebo-controlled study in rats.

MATERIALS AND METHODS

Design

A dose-escalating, placebo-controlled acute toxicity study was conducted in rats at Charles River Laboratories Preclini­cal Services Montreal (Senneville, Quebec, Canada), an animal laboratory registered with the U.S. Food and Drug Administra­tion (FDA) and the Canadian regulatory authorities. This study was conducted in compliance with current Good Laboratory Practices (cGLP).

Animal Welfare Statement

The study was approved by the Institutional Animal Care and Use Committee (IACUC).

Test Animals

Test animals were 96 rats (48 male, 48 female) (Charles River strain, derived from Sprague-Dawley; Charles River Canada, St. Constant, Quebec, Canada). Animals were approximately 12 weeks old and weighed 364 to 420 g (males) or 212 to 249 g (females) at the onset of treatment (day 1). Ani­mals were examined, quarantined, and reexamined prior to the study.

Animal Care and Handling

Animals were housed individually in stainless steel wire mesh-bottomed cages, accessed water ad libitum with an au­tomatic watering valve, and had free access to a standard labo­ratory diet (PMI Certified Rodent 5002; PMI Nutrition Interna­tional) except during designated procedures. Room temperature was 22°C ± 3°C with humidity 50% ± 20% and a 12-h light cycle.

Group Assignment

An acclimation period of 14 days was allowed between an­imal receipt and start of treatment. Prior to treatment, animals were weighed and randomly assigned to four treatment groups as described in Table 1. Randomization was by stratification using body weight as the parameter. Males and females were randomized separately. Animals in apparent poor health were not assigned to groups.

Drug Preparation and Testing

Proliferol (lidocaine hydrochloride 0.25%, dextrose 12.5%, glycerin 12.5%, and phenol 1.0% in sterile aqueous solution) was prepared according to current Good Manufacturing Prac­tices (cGMP) by a registered manufacturer (Bell-More Labs, Hampstead, MD). Testing was performed by a registered an­alytical laboratory (ACTA Laboratories, Foothill Ranch, CA) to assess drug ingredient concentrations, pH, osmolality, bacte­rial endotoxin, 5-hydoxymethylfufural (dextrose degradation), 2,6-dimethylalanine (lidocaine degradation), and heavy metals using methods adapted from the United States Pharmacopeia (United States Pharmacopeia 2003).

Dosages

Clinical studies with drugs similar to Proliferol for chronic low back pain have used doses of 10 to 30 ml; the midpoint (20 ml) was selected as the expected clinical dose and low-dose for this study (Dagenais, Haldeman, and Wooley 2005). Doses were based on a drug volume per bodyweight basis (ml/kg) because surface area is not a good indicator of spine size. The medium-dose was set at 5× the low-dose (equivalent to 100 ml in a 80-kg human) and the high-dose was set at 10× the low-dose (equivalent to 200 ml in a 80-kg human) (Table 1). Higher doses were not deemed feasible due to the likelihood of volume-related local tissue trauma.

Drug Administration

Intraligamentous spinal injections were not deemed feasible due to the size of the rat spine. As an alternative, intramuscular injections were given in the thoracolumbar region. For purposes of determining acute systemic toxicity, this route of administra­tion was deemed an appropriate surrogate. Animals were fasted overnight prior to dosing. On day 1 (hour 0), animals were anes­thetized with isoflurane gas and a bland lubricating ophthalmic agent was administered. The dorsum was trimmed of fur and the animal placed into a ventral recumbency for dosing. Prior to injection, the injection sites were cleaned with iodoret and alco­hol. Each animal was administered doses of the test or control article by multiple intramuscular injections in the thoracolum­bar paraspinal muscles bilaterally (volumes of 0.3 ml maximum per site) until the desired dosage was achieved. Areas bordering the needle (25-gauge) entry sites were marked with an indelible non-toxic marking pen immediately following dosing and daily thereafter. Additional clipping of the pelage was carried out to keep injection sites visible for necropsy.

Necropsy

Necropsies were conducted on day 2 for 48 animals (12 each from low-, medium-, high-dose Proliferol, and high-dose placebo control) and day 15 for the other 48 animals (12 each from low-, medium-, high-dose Proliferol, and high-dose placebo control). These time points were based on standard methodology for expanded acute toxicity studies, a previous pi­lot study, and discussions with the FDA regarding the protocol (Dagenais et al. 2006b). Rats were euthanized by exsanguination following isoflurane anesthesia.

Observations

Acute toxicity was assessed by clinical observations, clinical chemistry and hematology, urinalysis, as well as gross pathology and histopathology; local tolerance was assessed by injection site appearance and histopathology (see Tables 2 through 5). Analysis of blood and urine samples, specimens, and data was performed at the testing facility with the exception of urine lidocaine and phenol, which was performed at another clinical analytical laboratory (Pacific Toxicology Laboratories, Chatsworth, CA). Normal reference ranges were obtained from the test­ing laboratory and the literature (Swindle 1998; Swindle et al. 2003).

Clinical Observations

The animals were observed daily for the following: (1) mor­tality or moribundity; (2) detailed examination; (3) cage gait evaluation; (4) body temperature. Body weights were measured at randomization and days – 1,8, and 14. Prior to treatment and at study termination, a board-certified veterinary ophthal­mologist performed funduscopic (indirect ophthalmoscopy) and biomicroscopic (slit lamp) examinations in all animals.

Clinical Chemistry and Hematology

Tests outlined in Tables 2 and 3 were conducted at study termination (days 2 and 15). Food was removed overnight from all animals prior to testing but water was still available. Samples were collected in vacuum tubes containing EDTA or sodium citrate (for coagulation) from the abdominal aorta following isoflurane anesthesia.

Urinalysis

Samples were collected overnight prior to dosing. Immedi­ately following dosing on day 1, animals designated for day 14 necropsy were placed in metabolism cages where they were deprived of food but had access to water. Samples in this group were collected after 2, 8, and 24 h, and stored frozen (circa −20°C) until shipment on dry ice. Urine lidocaine was mea­sured by gas chromatography with flame ionization detection (GC-FID) and phenol was measured by high-performance liquid chromatography with ultraviolet detection (HPLC-U V). Prior to induction of anesthesia (isoflurane gas) for dosing, the animal received 1 ml of deionized water by oral gavage and the urinary bladder was emptied by digital manipulation. Other urine tests performed prior to study termination are shown in Table 6.

Gross Pathology and Histopathology

At study termination (days 2 and 15), samples were ob­tained from all major tissues and injection sites, including: adrenals; aorta (thoracic); bone and marrow (sternum); bone (femur); brain (cerebellum, forebrain, midbrain, and medulla oblongata); cecum; colon; duodenum; epididymides; esopha­gus; eyes; harderian glands; heart (including section of aorta); ileum; injection site (skin, subcutis, and muscle); jejunum; kid­neys; lacrimal glands; larynx (one level); liver (two lobes); lungs (two lobes); lymph nodes (mandibular, unilateral, mesenteric); mammary gland (inguinal); nasal cavity; optic nerves; ovaries; oviducts; pancreas; pharynx; pituitary; prostate; rectum; sali­vary gland (mandibular and unilateral); sciatic nerve; seminal vesicles; skeletal muscle; skin (inguinal); spinal cord (cervical); spleen; stomach; testes; thymus; thyroid lobes (and parathy­roids); tongue; trachea; urinary bladder; uterus (horns, body, and cervix); vagina; and zymbal gland. Tissues were prepared for histopathological examination by embedding in paraffin wax, sectioning, and staining with hematoxylin and eosin. A board-certified veterinary pathologist at the testing facility conducted the histopathological evaluation of the tissues.

Statistical Analysis

Means and standard deviations were calculated for contin­uous outcomes. Groups were compared with one-way analysis of variance (ANOVA), Dunnett’s test, Kruskal-Wallis test, or Dunn’s test based on distribution as determined by Levene’s test. Values below the lower limit of quantitation (LLQ) of <0.1 were assigned values of 0.05 (i.e., LLQ/2) for analysis. For each categorical parameter of interest, contingency tables were pre­pared and chi-square analyses were used to compare groups. For graphical representations, data were pooled among males and females within groups.

RESULTS

Clinical Observations

All clinical observations were within normal limits. There was no preterminal mortality, and no indication that the injection procedure or drug affected gait, ambulation, or general activity.

Clinical Chemistry

Findings of interest at 24 h included elevated aspartate aminotransferase (AST), alanine aminotransferase (ALT), and haptoglobin, with apparent dose-response relationships. At baseline and 14 days, all clinical chemistry values were within normal ranges. Results are presented in Table 2 and Figures 1 to 3.

Hematology

Findings of interest included elevated monocyte percentage at 24 h and elevated mean platelet volume (MPV) at 14 days, with apparent dose-response relationships. Other hematology parameters were within normal limits. Results are presented in Table 3.

Histopathology

There were no organ weight changes related to treatment. Gross findings that may have been treatment-related were dark­ness around injection sites for all Proliferol groups.

24 Hours

There was mild to moderate degeneration/necrosis of skele­tal muscle and minimal hemorrhage at the injection sites, with a mild increase in hemorrhage compared with control. Mild to slight degeneration/necrosis was present at the injection sites in control, low-dose, and medium-dose groups. Minimal to mod­erate degeneration/necrosis was present at the injection sites of the high-dose group. Similarly, low- and medium-dose Pro­liferol groups had minimal to slight subacute inflammation at the injection sites, whereas the high-dose Proliferol group had minimal to moderate changes. The control group had a mini­mal to slight mononuclear cell infiltrate at the injection sites. There were no other lesions anywhere else in the body (e.g., heart, liver, or lymph nodes) indicative of drug related systemic toxicity. Results are summarized in Table 4.

14 Days

There was regeneration of skeletal muscle, subacute to chronic inflammation, and fibrosis at the injection sites. There were only minimal changes of skeletal muscle regeneration and subacute to chronic inflammation at the injection sites of a few animals in the control group. Proliferol groups had mild to mod­erate regeneration of skeletal muscle and subacute to chronic inflammation, with a mild increase in the severity of skeletal muscle regeneration in the high-dose group. Also, minimal to slight fibrosis was present at the injection sites of Proliferol groups. A few animals in the Proliferol groups had residual minimal degeneration/necrosis at the injection sites. Other mi­croscopic findings seen in various organs and tissues were considered to be incidental or related to the experimental procedure. There were no other lesions anywhere else in the body indica­tive of drug related systemic toxicity. Results are summarized in Table 5.

Urinalysis

Urinalysis findings of interest at 24 hours included elevated urobilinogen and presence of blood in all dose groups compared to placebo (chi-square p < .05). For urine phenol and lidocaine, there were significant (ANOVA p < .05) group effects at each postdose time point. In general, urine lidocaine and phenol values peaked at 2 h post dose and tended to return to predose levels at 24 h for each group. There were also trends indicating an apparent dose-response relationship at 2 and 8 h post dose. This relationship disappeared at 24 h post dose, as all groups returned to predose levels. Results are summarized in Table 6 and Figures 4 and 5.

DISCUSSION

Results obtained in this study indicate that intramuscular Proliferol injections at 10× the clinical human dose result in localized acute inflammatory changes in soft tissues at the injection sites, with no evidence of systemic toxicity. These results are very similar to those reported in an earlier pilot study whose main findings were clinical chemistry, hematology, and urinalysis changes indicative of acute local inflammation; marked temporary increases in AST and ALT; and acute local inflammatory changes in the skin, subcutis, and muscle around the injection sites (Dagenais et al. 2006b). The present study methods differed slightly from the pilot study methods by (1) adding a high-dose saline control group to distinguish injection-related tissue trauma from drug-related local changes; (2) adding a 5 medium-dose group to study possible dose-response relationships; (3) increasing the high-dose from 5× to 10× the clinical human dose to establish a greater safety margin; (4) substituting haptoglobin for C-reactive protein as a marker for acute inflammation; (5) observing gait to assess changes related to increased pain; (6) measuring creatine kinase to assess muscle-related trauma; (7) increasing sample size from 1 to 12 per group; and (8) using a batch of Proliferol prepared according to Good Manufacturing Practices (GMP) rather than by a compound pharmacist.

The high-dose saline control group of the present study helped clarify that localized soft tissue changes around the injection sites (i.e., acute inflammation, necrosis, fibrosis) were partially attributable to the injection procedure. However, gross and microscopic histopathology observations in the Proliferol groups also suggest drug-related acute inflammatory changes of greater magnitude than saline controls. These changes are expected given the presence of hyperosmolar glucose and glycerin, as well as phenol. This precipitation of the acute inflammatory cascade at the injection site is perceived as beneficial because it precedes the desired connective tissue repair. A preliminary study using injections of this drug solution in humans with chronic low back pain reported histopathological changes that were consistent with those noted in the present study. Those findings include increased cellularity, increased active fibroblasts with plump and conspicuous nuclei, and a 60% increase in average fiber diameter in the posterior sacroiliac ligaments 3 months post treatment (Klein, Dorman, and Johnson 1989).

Despite histopathologic findings of skeletal muscle trauma and necrosis at the injection site, there were no elevations in creatine kinase (CK). This supports earlier reports that elevations in CK following skeletal muscle trauma and necrosis from intramuscular injection were only noted at 8 hours (Yagiela et al. 1981). Although CK is one of the preferred markers of skeletal muscle trauma, these findings suggest it is of limited use at 24 h and should be measured sooner to capture short-term changes that may otherwise be missed (Komulainen, Takala, and Vihko 1995).

In isolation, increases noted in ALT may have been attributed to this enzyme having been released from skeletal muscle due to trauma from the intramuscular injection procedure with a sizeable needle, as well as the large volume injected. When these findings are evaluated in conjunction with markedly elevated levels of AST, a more likely explanation was that of a temporary hepatic insult observed only at higher doses and which returned to normal at 14 days. This possibility would account for elevations in both ALT and AST, as well as elevated levels of urobilinogen at 24 h because hemolysis was not reported. It is also possible that elevated ALT was partially attributed to skeletal muscle trauma and temporary hepatic insult.

This possible temporary hepatic insult at higher doses did not appear to warrant concern given that there were no histopathologic lesions to the liver attributable to the drug solution, no irreversible alterations of hepatic clinical chemistry parameters, and no changes in general animal well-being as noted by clinical observations. Further studies may shed light on these hypotheses. Given the much lower current clinical dose for similar drug solutions, the risk of temporary hepatic insult in humans appears remote.

Significant increases were noted in serum haptoglobin levels at higher dose levels, confirming histopathologic findings of localized acute inflammatory soft tissue changes at the injection sites. These results also support the use of serum haptoglobin as a more sensitive marker of acute inflammation in rats than C-reactive protein (Giffen et al. 2003), which indicated neither changes from baseline to follow-up nor differences between dose groups in the previous pilot study (Dagenais et al. 2006b). The elevated monocyte percentage at 24 h is consistent with this finding and supports an acute inflammatory reaction at the injection sites. Though neutrophils were slightly elevated in higher dose groups, these changes were not statistically significant and not likely of toxicological significance; there were no other hematology findings. Decreases noted in albumin at 24 h in the high-dose group are also suggestive of acute inflammation, though changes were small and not statistically significant between groups. Exploration of additional markers of acute inflammation, including fibrinogen, may be warranted in future studies to confirm these findings.

Despite commonly observed temporary increases in local pain and discomfort following injections with this drug solution in humans—which could negatively impact gait—there were no such changes even at 10× the clinical human dose. In addition, there were no signs of overt pain behavior. Although interpretation of these findings is challenging given that test animals were quadrupeds rather than bipeds, it appears that intramuscular administration of Proliferol in rats at 10× the human clinical dose was well tolerated.

A preliminary pharmacokinetic evaluation of Proliferol was undertaken by assessing urine excretion of two ingredients, phenol and lidocaine. An apparent dose-response increase in urine phenol and lidocaine levels was observed following intramuscular injection of Proliferol at 3 doses (1×, 5×, and 10× of the proposed human dose) and placebo control. Results also indicate that urine phenol and lidocaine levels are highest after 2 h, decrease by approximately half of their maximal concentration after 8 (lidocaine) or 12 (phenol) h, and are no longer detected after 24 h. This suggests that Proliferol is rapidly eliminated following intramuscular injection. Glucose was not detected in routine urinalysis, indicating that levels present in Proliferol are easily metabolized and not excreted. Glycerin was not measured because it is metabolized to glucose and triglycerides, which were slightly elevated in high-dose groups.

In conclusion, intramuscular injections of Proliferol in rats of up to 10× the human clinical dose, a drug solution commonly used in prolotherapy that contains lidocaine hydrochloride 0.25%, dextrose 12.5%, glycerin 12.5%, and phenol 1.0%, elicited localized soft tissue changes consistent with acute inflammation at the injection site, and no indications of systemic toxicity. Additional research in nonrodents should be undertaken to confirm these results.