Borneol, a messenger agent, improves central nervous system drug delivery through enhancing blood–brain barrier permeability: a preclinical systematic review and meta-analysis

Abstract To achieve sufficient blood–brain barrier (BBB), penetration is one of the biggest challenges in the development of diagnostic and therapeutic for central nervous system (CNS) disorders. Here, we conducted a systematic review and meta-analysis to assess the preclinical evidence and possible mechanisms of borneol for improving co-administration of CNS drug delivery in animal models. The electronic literature search was conducted in six databases. Fifty-eight studies with 63 comparisons involved 1137 animals were included. Among 47 studies reporting the assessments of CNS drug concentration, 45 studies showed the significant effects of borneol for improving CNS drug delivery (p<.05), whereas 2 studies showed no difference (p>.05). Nineteen comparisons showed borneol up-regulated BBB permeability (p<.05) using brain EB content (n = 8), Rh 123 content (n = 4), brain imaging agent content (n = 2), brain water content (n = 1) and observing ultrastructure of BBB (n = 4), whereas three studies showed no difference or unclear results. Seven studies reported the safety, in which one study showed borneol was reversible changes in the BBB penetration; six studies showed borneol did not increase co-administration of blood drugs concentration of peripheral tissues (p > .05). Effects of borneol are closely associated with inhibition of efflux protein function, releasement of tight junction protein, increasement of vasodilatory neurotransmitters, and inhibition of active transport by ion channels. In conclusion, borneol is a promising candidate for CNS drug delivery, mainly through mediating a multi-targeted BBB permeability.


Introduction
A key obstacle for therapeutic drugs administered for central nerve system (CNS) disease is passage across the blood-brain barrier (BBB) (Abbott, 2013). The BBB is a specialized nonpermeable barrier constituted by endothelial cells, a basal lamina and astrocytic endfeet (Zlokovic, 2008). It serves a predominant role in regulating supply of essential nutrients to the brain as well as protecting the CNS from many potentially harmful compounds (Abbott et al., 2010). The property of selective impermeable BBB is mainly due to the presence of tight junctions between adjacent endothelial cells and the existence of various BBB transporters, e.g. efflux transporters P-glycoprotein (P-gp). The tight junctions are against the access of about 100% of large-molecule neurotherapeutics and $98% of all small-molecule drugs to the brain (Pardridge, 2005). The BBB transporters are against the accumulation of a wide range of drugs in brain (Demeule et al., 2002). Thus, the BBB maintains the brain homeostasis and also inhibits the entry of potentially useful diagnostic and therapeutic agents, which consequently restricts the therapeutic effects of majority of drugs on many CNS disorders (Abbott et al., 2006).
The past 30 years have seen a great deal of research on the CNS drug delivery, and several strategies have been tried to deal with the problem (Banks, 2016). For example, highly invasive strategies, i.e. intracerebral or intracerebroventricular administration are useful for local CNS delivery in specific cases e.g. in well-defined tumors, but they are risky, costly, and of limited value for the administration of therapeutic agents that are directed toward less localized diseases such as diffused tumors, Alzheimer's disease, and multiple sclerosis (Garcia et al., 2005). Furthermore, higher concentrations of drug facilitate entry, but efficacy is limited by dose-dependent toxicity of peripheral tissues (Banks, 2016). What is more, approaches that disrupt an intact BBB in an attempt to let in a candidate drug also let in circulating substances that are normally excluded by the BBB and can be quite toxic to the CNS (Kroll & Neuwelt, 1998). Thus, numerous intravascular drugs delivery strategies which consider BBB as a therapeutic target have been proposed gradually and tested in hope of enhancing BBB penetration instead of disrupting BBB to achieve a widespread transport of the infused drug across the whole brain parenchyma (Tosi et al., 2008). Up to now, a number of intravascular strategies have been explored to improve the transport of drug across BBB, such as osmotic and chemical modifications of BBB, enhanced transcellular transport, nanoparticle carriers, and cell-based drug delivery (Hersh et al., 2016). This is a promising but difficult area of drug development, as specific features, advantages, and limitations in every strategy (Hersh et al., 2016), and few drugs have been successfully applied to the clinic . This complexity confounds simple strategies for drug delivery to the CNS, but provides a wealth of opportunities and approaches for drug development (Banks, 2016).
Borneol, highly lipid-soluble bicyclic terpene chemicals extracted from Cinnamomum camphora (L.) Presl. and Blumea balsamifera (L.) DC. or chemically transformed on the basis of camphor and turpentine oil (State Pharmacopoeia Committee, 2010), is widely used as a messenger drug in many traditional Chinese herbal prescriptions such as Angong Niuhuang pill, a well-known formula for treating stroke . According to traditional Chinese medicine (TCM) Emperor-Minister-Assistant-Courier theory, this principle guides the combination of multiple herbal medicines in a specific manner when creating TCM compound prescriptions. Borneol is classified as a 'Courier herb' that guides the herbs upward to target organ, especially in the upper part of the body, such as the brain. This studies showed that borneol is not only an effective penetration enhancer through corneal (Yang et al., 2009), intestinal mucosa , and nasal cavity mucosa (Lu et al., 2011) but also an effective BBB penetration enhancer for a greater access of drug to the brain . The increased CNS concentrations of carbamazepine and valproate after the co-administration of borneol in epileptic patients with few side effects have been reported in clinical trials (Xu et al., 2016;Armulik et al., 2010). However, insufficient evidence and unknown mechanism limited the application of borneol in clinic . Thus, we conducted a preclinical systematic review to provide the preclinical evidence and possible mechanisms of borneol on upregulation of BBB permeability to enhance CNS drug concentrations.

Search strategy
The systematically electronic literature search was conducted via PubMed, Chinese National Knowledge Infrastructure, VIP Database, Wanfang database, and Chinese Biomedical Database from their inceptions to December 2017. The search terms were as follows: 'borneol OR camphol' AND 'blood brain barrier' in Chinese or in English. All searches were limited to animal studies.

Eligibility criteria
Studies of borneol for CNS drug delivery through enhancing BBB permeability in vivo were included. There was no restriction on animal species or publication status. Eligibility criteria were: (Abbott, 2013) borneol for animal, regardless of its mode, dosage and the administration frequency; (Zlokovic, 2008) the primary outcome measures were the co-administration of drug concentrations in CNS, and the second outcome measures were the safety of borneol, the various indexes of BBB permeability, and possible mechanisms of borneol for enhancing BBB permeability; (Abbott et al., 2010) interventions for control group were isasteric and nonfunctional liquid (normal saline) or no treatment. Exclusion criteria were predefined as follows: (Abbott, 2013) case reports, reviews, abstracts, news, comments, editorials, and in vitro studies; (Zlokovic, 2008) compared with medicine or another agent with potential similar effect; (Abbott et al., 2010) was not tested on the primary and/or second outcome measures; (Pardridge, 2005) lack of control group; (Demeule et al., 2002) duplicate publication.

Data extraction
Two authors independently reviewed each included study and extracted following aspects of details: (Abbott, 2013) name of first author, year of publication and method of anesthesia and/or model; (Zlokovic, 2008) details (species, number, sex, and weight) of animals for each study; (Abbott et al., 2010) the use of anesthesia in the experiment and the methods to establish animal models; (Pardridge, 2005) the information on the method of administration was obtained from both treatment and control group including drug, dose, mode and frequency; (Demeule et al., 2002) the outcome measures and samples for individual comparison were included. A comparison was defined as the qualitative and/ or quantitative assessments of co-administration of drug concentrations in CNS and/or the safety of borneol and/or the various indexes of BBB permeability in treatment and corresponding control group after the administration of borneol or vehicle with a given dose, mode, and frequency. In case of lack of vehicle group, the group receiving no adjunct intervention was used as control group for individual comparison. If a drug concentration was used for outcome assessment, both the drug and the method of drug administration were obtained. All available data from quantitative assessments of primary and second outcomes were extracted for every comparison including mean outcome and standard deviation (Abbott et al., 2006). The efficacy result was summarized as increased or decreased according to whether a significantly increasing or decreasing outcomes in each study. If there was no statistical difference of effects of borneol between treatment and control groups, the efficacy results were summarized as no difference. In instances of absence of statistical analysis within comparison as well as available original data, the efficacy result of the comparison was listed as "increased?" or "decreased?"

Statistical analysis
The statistical analysis was conducted via RevMan version 5.3 software in Copenhagen, Denmark. To estimate the effect of borneol on CNS drug delivery and/or BBB permeability across studies, a summary statistic was calculated for each comparison with 95% confidence intervals by using the random effects method. When the outcome measurements in all included studies in meta-analysis were based on the same scale, weighted mean difference (WMD) was calculated as a summary statistic. On the contrary, when the same outcome measurements were measured in a variety of ways across studies in meta-analysis, standardized mean differences (SMD) was used as a summary statistic. Heterogeneity between study results was investigated based on a standard chi-square test and I 2 statistic. A probability value .05 was considered statistically significant.

Quality of included study
The quality scores of studies included varied from 1 to 5 out of 10 points with the average of 2.8. Among them, 1 study scored 1 point; 22 studies scored 2 points; 24 studies scored 3 points; 8 studies scored 4 points; 3 studies scored 5 points (Table 2). Forty-seven studies were peer-reviewed publication and 11 studies were Master's thesis or PhD thesis. Six studies described the control of temperature. Forty-seven studies declared the random allocation. Forty-five studies described the use of anesthetic without significant intrinsic neuroprotective activity. Sixteen studies stated the compliance with animal welfare regulations. Three studies described the application of animal or model with relevant comorbidities. None of the studies included reported the masked conduct of experiments, the blinded assessments of outcome, a sample size calculation or a statement of potential conflict of interests.

Co-administration of drug concentrations in CNS
Forty-seven studies reporting the assessments of co-administration of drug concentrations in CNS, of which 45 studies showed the significant effects of borneol for improving CNS drug delivery and 2 studies showed no difference (Chen, 2005;. Among the 45 studies, several main categories of drugs were reported, including antineoplastic drugs, antibiotics, antiviral drugs, drugs for epileptic, Parkinsonism and cognition. Some Chinese herbal medicines also were mentioned. Eight types of the drugs were reported more than once. There studies investigated the effect of borneol on tetramethylpyrazine concentration-curve in brain tissue Xiao & Ping, 2009) and in CSF ; three studies Jia et al., 2004;Yin et al., 2017) on the brain concentration of cisplatin; two studies on the brain concentration ) and on the CSF concentration of methotrexate ; two studies Zhang et al., 2015) on the brain concentration of Kaempferol; two studies Zhang et al., 2011) on the brain concentration and one study  on the CSF to serum concentration ratio of carbamazepine over time; three studies Zhang et al., 2007; on CSF concentrationcurve of valproate; two studies Yu et al., 2012) on the main pharmacokinetic parameters of geniposide in brain tissue; two studies on the brain concentration  and the CSF to serum concentration ratio  of Salvia miltiorrhiza over time (Table 3).

BBB permeability and meta-analysis
Nine studies Lin et al., 2003;Zhang et al., 2005;Zhu, 2009;Yu et al., 2011;Wu et al., 2011;Huang et al., 2013;Yin et al., 2017) used EB content as outcome measures to test the BBB permeability and involved following 11 comparisons: 8 comparisons Lin et al., 2003;Zhang et al., 2005;Zhu, 2009;Yu et al., 2011;Wu et al., 2011;Huang et al., 2013;Yin et al., 2017) with increased effects (p < .05), 1 comparison  with no difference (p > .05), and 2 comparisons  listed as "increased?" without data. 28,31,53,59,60,67,80) comparisons with available data showed significant effects of borneol for increasing brain EB content compared with control (n ¼ 141, SMD 5.85, 95% CI: 3.56 $ 8.14, p < .00001). There was high heterogeneity among these 8 comparisons (v 2 ¼ 87.54, p < .00001, I 2 ¼ 92%). Thus, subgroup analysis was followed according to stratification on animal species, the frequency of administration, the mode of application, the dose of administration and the instrument used for quantification of brain EB content. In the subgroup analyses of these factors, the effect size of rat species was larger than other two animal mice and guinea pigs species (SMD ¼ 11.59 vs. SMD ¼ 4.27 vs. SMD ¼ 4.79, Figure 2(A)). The effect size of single administration animals was greater than successive administration animals (SMD ¼ 9.11 vs. SMD ¼ 2.72, Figure 2(B)). The mode of application showed great discrepancy in the overall effect of outcome measure, which the administration by acupoint injection with only scale of 7.2% weight accounted for greater effect size than by intranasal administration and gavage (SMD ¼ 17.55 vs. SMD ¼ 4.79 vs. SMD ¼ 4.77, Figure 2(C)).
The effect size was greater in animals using fluorescence microscopy than in animals using other quantified method, including UV spectrophotometer, fluorescence spectrophotometer, ELISA instrument (Figure 2(D)). The group that the therapeutic dose of borneol larger than 0.5 g/kg showed greater effect size than the group with 0.5 g/kg or less dose (SMD ¼ 9.37 vs. SMD ¼ 3.93, Figure 2(E)). The lower quality studies exhibit larger effect size than the higher ones (SMD ¼ 9.38 vs. SMD ¼ 4.68, Figure 2(F)). Four studies Yu et al., 2013;Yu et al., 2015; used Rh 123 content as A: peer-reviewed publication; B: monitoring of physiological parameters such as temperature; C: random allocation; D: blinded conduct of the experiments; E: blinded assessment of outcome; F: use of anesthetic without significant intrinsic neuroprotective activity (e.g. ketamine); G: animal and/or model (brain tumor model, epilepsy, intracranial infection, cognitive dysfunction or Parkinson); H: sample size calculation; I: compliance with animal welfare regulations; J: statement of potential conflict of interests.
outcome measures to test the BBB permeability, after removing 1 study  for concentration-curve of Rh 123, metaanalysis of three studies  indicated that borneol can improve Rh123 concentration in CNS significantly compared with control (n ¼ 30, SMD 1.48, 95% CI: 0.89 $ 2.08, p < .00001). There was low heterogeneity among the three included studies (v 2 ¼ 3.72, p ¼ .16, I 2 ¼ 46%) ( Figure  3). Compared with controls, two studies ; Table 3. The classification of drugs transferred into the brain.
Zhang, 2011) showed significant effects of borneol for increasing brain imaging agent entering the brain (p < .05) but failed to obtain primary data for poor analysis, one study  for increasing brain water content (p < .05), four studies Ge et al., 2008;Yu et al., 2011Yu et al., , 2013 for increasing the opening effects of the ultrastructure of BBB (p < .05).

The safety of co-administration of borneol
Six studies Yu et al., 2012;Cao, 2013;Diao et al., 2013;Xin et al., 2014) indicated that the increased effects of borneol on brain or CSF drug concentration were accompanied by the absence of an increase in the blood drug concentration. One study (Ge et al., 2008) reported that the opening of BBB by borneol has been found to be reversible and physiological in accordance with the ultrastructure assessments of BBB, which could last up to 8 h after its intragastric administration in rats.

Summary of evidence
This is the first preclinical systematic review to determine the effects of borneol on CNS drug delivery in animal models.
Fifty-eight with 1137 animals were selected. The quality of studies included was generally medium. The evidence available from this study showed that the co-administration of borneol is a promising candidate for CNS drug delivery. The effects of borneol are closely associated with the inhibition of efflux protein function, releasement of tight junction protein, increasement of vasodilatory neurotransmitters, and inhibition of active transport by ion channels.

Limitations
Our study only included two animal species, rodent, and rabbit, which may potentially impose restrictions on the promotion of the findings. The significant heterogeneity across studies indicates that conclusions should have been treated more cautious. The methodological quality of studies included was generally moderate, which is an inherent drawback in the primary study. It was indicated that a lack of blinding outcome assessments attributed to a 27% overestimation of the mean reported effect size (Holman et al., 2015). No study reported the data on a sample size calculation, which may inflate the reported effect size. Therefore, the results in this study should be interpreted with caution.

Implications
In this study, the findings showed the enhanced penetration of a variety of drugs acting on the CNS and increased BBB permeability of EB and Rh 123 after the co-administration of borneol. Thus, we proposed accordingly the co-administration of borneol as a potential approach for effective brain drug delivery with several advantages. First, the administration of borneol is noninvasive and allows for repeated applications by gavage, intravenous injection, and nasal administration. Second, the increased effects of borneol on brain or CSF drug concentration were accompanied by the absence of an increase in the blood drug concentration Yu et al., 2012;Cao, 2013;Diao et al., 2013;Xin et al., 2014), which indicated that the co-administration of borneol did not increase the risk of peripheral adverse effects. Third, the opening of BBB by borneol has been found to be reversible and physiological in accordance with the ultrastructure assessments of BBB, which could last up to 8 h after its intragastric administration in rats (Ge et al., 2008) and did not cause an up-regulation of inducible nitric oxide synthase (Baoshe & Qi de, 2002), the over-expression that always occurred in the presence of pathological processes, e.g. Hypoxia (Robinson et al., 2011). Thus, the co- administration of borneol may be a safe and promising strategy for effective BBB penetration enhancer for CNS drug. The evidence of mechanisms available from this study showed that borneol enhanced BBB permeability largely through inhibiting efflux protein function, releasing tight junction protein, increasing vasodilatory neurotransmitters, inhibiting active transport by ion channels. Moreover, some studies Li et al., 2012) reported that borneol can increase the levels of excitatory amino acid greater than the levels of inhibitory amino acids increased in the whole brain, leading to a transient elevation in the excitation ratio, which was conjectured as a reason of the transient and reversible effects of borneol on enhancing BBB permeability. Thus, borneol for opening BBB permeability transiently and reversibly depended on multi-targeted mechanisms.

Conclusions
Our findings indicate that borneol is a multi-targeted BBB permeability mediator, suggesting that the co-administration of borneol is a promising candidate for CNS drug delivery. The effects of borneol are closely associated with the inhibition of efflux protein function, the releasement of the tight junction protein, increasement of vasodilatory neurotransmitters, and inhibition of active transport by ion channels.